KR20160117496A - Culture medium composition - Google Patents

Abstract

The present invention provides a method of culturing cells and / or tissues comprising culturing cells and / or tissues in a suspended state by using a culture medium composition having an effect of preventing sedimentation of cells and / or tissues, By maintaining the cells and / or tissues substantially without substantially increasing the viscosity of the solution by nanofibers added to and dispersed uniformly in a liquid medium or the like.

Description

[0001] CULTURE MEDIUM COMPOSITION [0002]

The present invention relates to a culture composition for cultivating animal and plant cells and / or tissues, particularly in a three-dimensional or suspended state, using nanofibers such as polysaccharides with enhanced dispersibility in water, and uses thereof.

BACKGROUND ART [0002] In recent years, techniques for growing or maintaining various organs, tissues, and cells that play different roles in plants and animals in vitro have been developed. The propagation or maintenance of organs and tissues in vitro is referred to as organ culture and tissue culture, respectively, and the propagation, differentiation or maintenance of cells isolated from organs or tissues in vitro is called cell culture. Cell culture is a technique for proliferating, differentiating, or maintaining isolated cells in vitro in a medium, and is indispensable for detailed analysis of the functions and structures of various organs, tissues and cells in vivo. In addition, the cells and / or tissues cultured by the above techniques can be used for evaluation of efficacy and toxicity of chemical substances and pharmaceutical products, mass production of useful substances such as enzymes, cell growth factors, antibodies, Regenerative medicine that replenishes tissues and cells, breeding of plants, production of genetically engineered products, and the like.

Animal-derived cells are broadly divided into non-adherent and adherent cells based on their characteristics. Non-adherent cells are cells that do not require a scaffold for growth and proliferation, and adherent cells are cells that require a scaffold for growth and proliferation. Most of the cells that constitute the living body are the adherent cells of the latter. As a method of culturing adherent cells, there are known single layer culture, dispersion culture, forced culture, micro carrier culture, sphere culture and the like.

The monolayer culture is a method of culturing a target cell as a monolayer by using a culture container made of glass or a synthetic polymer material subjected to various surface treatments or a support cell called a feeder cell as a scaffold, . For example, it is possible to apply various surface treatments (plasma treatment, corona treatment, etc.), coating with cell attachment factors such as collagen, fibronectin, polylysine and the like, or polystyrene such as pre- A culture method using culture containers of various shapes or characteristics has been developed. However, in the monolayer culture, since the two-dimensional culture environment is completely different from the in vivo environment, the cell can not maintain the specific function possessed in the living body for a long time, and the cell can not reconstruct a tissue similar to the living body , Which is a problem in that it is not suitable for mass culture of cells because the number of cells per a certain area is limited (Patent Document 1). In addition, the method of culturing a desired cell on a feeder cell sometimes faces a problem in separation of a feeder cell and a desired cell (Non-Patent Document 1).

Dispersion culture comprises the steps of seeding cells in a medium and stirring the culture medium in a culture vessel to which the surface treatment for inhibiting cell attachment is applied to inhibit adhesion of the cells to the culture vessel, It is a method of culturing cells. However, the adherent cells cultured in this manner can not be attached to the scaffold and therefore can not be applied to cells that essentially require adherence to the scaffold for cell proliferation. In addition, since the cells can not exhibit their original cell functions due to continuous destruction by shearing force, functional cells can sometimes not be cultured in large quantities (Non-Patent Document 2).

Formed culture is a method of culturing cells by embedding and fixing cells in a solid or semi-solid gel matrix such as agar, methylcellulose, collagen gel, gelatin, fibrin, agarose, alginate and the like. This method makes it possible to cultivate the cells three-dimensionally close to the living body, and since the gel substrate itself sometimes promotes cell proliferation and differentiation, Cells can be cultured at high density (Patent Documents 2 and 3). Furthermore, a cell culture method comprising forming microcapsules having a size of 100 to 300 mu m by embedding cells in a gel matrix and culturing the cells in an aqueous solution medium while dispersing the microcapsules has also been developed (Non-Patent Document 3) . However, this method is incapable of continuously observing the cultured cells unless the visible light is transmitted through the gel matrix. Since the medium containing the gel matrix and the microcapsules have high viscosity, For example, collagenase treatment in the case of collagen gel), and the like, and it is difficult to exchange the culture medium required for long-term culture. BACKGROUND ART [0002] Recently, techniques for enabling cell recovery from a gel substrate by treatment such as heat and shearing force have been developed. However, heat, shear force and the like may adversely affect the cell function, and the safety of the gel substrate against the living body has not yet been clarified (Patent Documents 4 and 5, Non-Patent Documents 4, 5, 6 and 7). In addition, sol foods that are uniformly dispersed and suspended in a foodstuff are prevented from being precipitated and suspended in foods such as fruits and vegetables, which have been cut into small pieces, and have been developed in the field of foods. However, sol foods do not consider recovering the dispersed granular food, and it has not been tested whether cells and tissues can be suspended or cultured (Patent Document 6). It is known that gellan in an aqueous solution gels by the action of calcium ions to form a microstructure (Non-Patent Document 8).

Microcarrier culture is carried out by growing cells in a monolayer on the surface of microparticles slightly larger than water (hereinafter also referred to as microcarriers), stirring the microparticles in a culture vessel such as a flask, and culturing the cells in a suspended state Method. Typically, the microcarriers used in the method are spherical particles having a diameter of 100 to 300 mu m, a surface area of 3000 to 6000 cm < ² > / g and a specific gravity of 1.03 to 1.05, and are composed of dextran, gelatin, alginic acid or polystyrene. Charged groups such as collagen, gelatin or dimethylaminoethyl may also be added to the surface of the microcarrier to facilitate attachment of the cells. This method is applied to the mass culture of cells because the cultivation area can be greatly increased (Patent Documents 7 and 8). However, it is difficult to uniformly adhere target cells to all the microcarriers, and problems such as desorption of cells from the microcarriers due to shear force during stirring, damage to cells, and the like arise (Non-Patent Document 9).

Spear culture is a culture method comprising culturing an agglomerate (hereinafter also referred to as a spear) consisting of several hundreds to several hundreds of cells as desired and culturing the agglutinates while allowing to stand or shake in the medium. It is known that sphere has a high cell density, can reconstitute cell-cell interactions and cell structure close to that of the in vivo environment, and can be cultured while maintaining cell function for a longer period of time than the single layer culture and dispersion culture method (Non-Patent Documents 10 and 11). However, if the size of the spear is too large, the spear culture can not form a large sphere because it is difficult to supply the nutrients inside the spear and to exhaust waste products. In addition, since the formed spear needs to be cultured in a dispersed state in the lower part of the culture container, the number of spores per given volume can not be easily increased and is not suitable for mass culture. In addition, as a method of forming a sphere, there can be used a suspension culture method such as hanging drop culture, culture on a cell non-adherent surface, culture in a microwell, rotation culture, culture using a cell scaffold, centrifugal force, And the like. However, such a method has problems such as complicated operations, difficulty in spear recovery, difficulty in size control and mass production, uncertain effects on cells, and the need for special dedicated containers and devices 9).

On the other hand, the plant, organs, and union tissues such as cells, protoplasts without cell walls or leaves, stems, roots, growth points, seeds, pears, pollen, etc., on the other hand, . Using the plant tissue and cell culture techniques, it has become possible to improve the variety of plants and produce useful components. As a method for mass-proliferating plant cells and tissues in a short period of time, there has been known a method of suspending plant cells and tissues in a liquid medium (Non-Patent Document 12). In order to achieve satisfactory proliferation thereof, it is important to supply sufficient oxygen, maintain a uniform mixed state, and prevent cell damage. The oxygen supply to the culture medium and the suspension of cells and tissues can be performed by a combination of aeration and mechanical agitation or by aeration alone. The former may result in poor proliferation due to damage to cells and tissues due to agitation and the latter may be difficult to maintain uniform mixing conditions in high density cultures even though cell or tissue shear is small, There is a problem such that the precipitation efficiency is lowered by forming the precipitate.

Japanese Patent Application Laid-Open No. 2001-128660 Japanese Laid-Open Patent Publication No. 62-171680 Japanese Laid-Open Patent Publication No. 63-209581 Japanese Laid-Open Patent Publication No. 2009-29967 Japanese Laid-Open Patent Publication No. 2005-60570 Japanese Patent Application Laid-Open No. 8-23893 Japanese Patent Application Laid-Open No. 2004-236553 International Publication No. 2010/059775 Japanese Laid-Open Patent Publication No. 2012-65555

Klimanskaya et al., Lancet 2005, 365: 1636-1641 King et al., Curr Opin Chem Biol. 2007, 11: 394-398 Murua et al., J. of Controlled Release 2008, 132: 76-83 Mendes, Chemical Society Reviews 2008, 37: 2512-2529 Moon et al., Chemical Society Reviews 2012, 41: 4860-4883 Pek et al., Nature Nanotechnol. 2008, 3: 671-675 Liu et al., Soft Matter 2011, 7: 5430-5436 Perez-Campos et al., Food Hydrocolloids 2012, 28: 291-300 Leung et al., Tissue Engineering 2011, 17: 165-172 Stahl et al., Biochem. Biophys. Res. Comm. 2004, 322: 684-692 Lin et al., Biotechnol J. 2008, 3: 1172-1184 Weathers et al., Appl Microbiol Biotechnol 2010, 85: 1339-1351

DISCLOSURE OF THE INVENTION An object of the present invention is to solve the problems of the prior art described above, and it is an object of the present invention to provide a culture composition for cultivating cells and / or tissues of animals and plants, in particular three-dimensional or suspended state, and cells and / And a method of culturing the tissue.

As a result of intensive studies, the inventors of the present invention have found that, by mixing nanofibers composed of polysaccharides such as cellulose and chitin into a liquid medium, suspension culture of animal and plant cells and / or tissues can be carried out without substantially increasing the viscosity of the liquid medium And that the cell growth activity is promoted by culturing the cells using this culture medium composition. In addition, not only water-insoluble polysaccharides such as cellulose but also water-soluble polysaccharides such as deacylated gellan gum form fibrous-like structures in a liquid medium, and this does not substantially increase the viscosity of the liquid medium, And / or allowing the suspended culture of the tissue to remain in a stationary state. Furthermore, they have further found that they can easily recover the cultured cells and / or tissues from the medium composition. On the basis of the above findings, further studies were conducted to complete the present invention.

That is, the present invention is as follows:

[1] A medium composition capable of culturing cells or tissues in a suspension state, comprising a nanofiber.

[2] The medium composition according to the above [1], which permits exchange of the culture medium composition during culture and recovery of cells or tissues after completion of culture.

[3] The medium composition according to the above [1], wherein any of temperature change, chemical treatment, enzyme treatment and shearing force is not required at the time of cell or tissue recovery.

[4] The medium composition according to the above [1], wherein the viscosity is 8 mPa · s or less.

[5] The method according to the above [1], wherein the nanofiber has an average fiber diameter of 0.001 to 1.00 μm and an average fiber length (L) to an average fiber diameter (D) of 2 to 500 Composition.

[6] The medium composition according to the above [1], wherein the nanofiber is composed of a polymer compound.

[7] The culture medium according to [6], wherein the polymer compound is a polysaccharide.

[8] The pharmaceutical composition according to the above [7]

Water-insoluble polysaccharides selected from the group consisting of cellulose, chitin and chitosan; or

There may be mentioned hyaluronic acid, gellan gum, deacylated gellan gum, rhamzaen gum, dituan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectic acid, heparan sulfate, heparin, Wherein the water-soluble polysaccharide is selected from the group consisting of alginic acid, alginic acid, and salts thereof.

[9] The culture medium according to the above [8], wherein the polysaccharide comprises cellulose or chitin.

[10] The medium composition according to the above [9], wherein the nanofibers are obtained by pulverization.

[11] The culture medium for cell culture according to any one of [1] to [10] above.

[12] The medium composition according to the above [11], wherein the cells are adherent cells or non-adherent cells.

[13] The medium composition according to the above [12], wherein the adhered cell is a sphere.

[14] A culture medium according to any one of [1] to [13], and a cell or tissue culture comprising cells or tissues.

[15] A method for culturing a cell or tissue, which comprises culturing a cell or tissue in the medium composition according to any one of [1] to [13].

[16] A method for recovering a cell or tissue, which comprises separating a cell or tissue from the culture according to [14] above.

[17] The method according to the above [16], wherein the separation is performed by centrifugation.

[18] A method for producing a sphere, comprising culturing adherent cells in a medium composition according to any one of [1] to [13].

[19] A culture medium additive for preparing the culture medium composition according to any one of [1] to [13], wherein the culture medium comprises the nanofiber or the water-soluble polymer compound constituting the nanofiber.

[20] A method for producing a culture medium composition, which comprises mixing a culture medium according to [19] with a culture medium.

[21] A process for producing a culture medium according to any one of [1] to [13], which comprises mixing a culture medium with a water-soluble polymer compound constituting the nanofiber or the nanofiber, Way.

[22] A method for preserving cells or tissues, comprising preserving cells or tissues in the culture medium composition according to any one of [1] to [13].

[23] A method for transporting cells or tissues, comprising transporting cells or tissues in the culture medium composition according to any one of [1] to [13].

[24] A method for growing a cell or a tissue, which comprises culturing a cell or tissue in the medium composition according to any one of [1] to [13].

[25] A method for subculturing adherent cells, comprising the steps of:

(1) suspending adherent cells in the culture composition according to any one of [1] to [13] above; And

(2) A method for culturing a cell culture comprising the steps of: (i) cultivating a culture comprising adherent cells obtained by the suspension culture of step (1) (Ii) adding a culture medium containing the adherent cells obtained by the suspension culture of step (1) to the culture medium according to any one of the above [1] to [13] Adding all or part of the water.

[26] A method for propagating adherent cells, which comprises suspending adherent cells in a culture medium containing chitin nanofibers while attaching the adherent cells to the chitin nanofibers.

[27] The method according to the above 26, wherein the content of chitin nanofibers in the culture medium composition is 0.0001% (weight / volume) to 0.1% (weight / volume).

The present invention provides a medium composition containing nanofibers, in particular, nanofibers composed of polysaccharides. Using the culture medium composition, the cells and / or tissues can be cultured in a suspension state without any operation such as shaking, rotation or the like, which may cause damage and loss of cells and tissues. Further, using the above-mentioned culture medium composition, the medium can be easily exchanged during culturing, and the cultured cells and / or tissues can also be easily recovered. The present invention can be applied to cells and / or tissues collected from animals or plants, and the desired cells and / or tissues can be mass-produced without impairing their function. Cells and / or tissues obtained by the culture method can be used for the evaluation of drug efficacy and toxicity of chemical substances and medicines, mass production of useful substances such as enzymes, cell proliferation factors, antibodies, organs and tissues And regenerative medicine for supplementing cells.

Further, since the culture medium composition of the present invention can keep the cells or tissues in an environment close to the biological environment, it is useful for preserving and transporting cells and tissues. For example, when cells are adhered and cultured on a plate, and the plate is transported as it is, vibration during transportation may cause the cells to peel off the plate, thus deteriorating the inherent functions of the cells. However, in the culture medium composition of the present invention, the nanofibers form a three-dimensional network, and by supporting the cells, the cells can be maintained in a suspended state. Therefore, it is possible to avoid damage to the cell such as peeling from the plate, which is generated by the vibration during transportation, and the cell can be preserved and transported while maintaining the original function of the cell.

Fig. 1 is a diagram showing that, when a sphere of HepG2 cells is cultured in a medium composition, the sphere can be uniformly dispersed and cultured in a suspended state.
[Fig. 2] is a figure showing that, when a sphere of HeLa cells is cultured in a medium composition, the sphere can be uniformly dispersed and cultured in a suspended state.
[Fig. 3] is a diagram showing that when the sphere of HeLa cells is cultured and observed under a microscope, association of spears can be suppressed compared to conventional media.
[Fig. 4] is a diagram showing that HepG2 cells can be proliferated on a micro carrier when a microcarrier on which HepG2 cells are adhered is cultured in a medium composition.
[Fig. 5] is a figure showing that when the sphere of HeLa cells is added to the culture composition, the sphere is uniformly dispersed and in a suspended state.
[Fig. 6] is a figure showing that a sphere of HeLa cells can be formed in a medium composition.
7 is a view showing a film as one embodiment of the structure, wherein the concentration of the deacylated gellan gum in the culture medium composition was 0.02% (weight / volume).
[Fig. 8] is a figure showing that a sphere of HepG2 cells can be formed in a medium composition.
[FIG. 9] is a diagram showing the suspension state of a laminin-coated GEM adhered with HepG2 cells when cultured in a medium composition.
[Fig. 10] is a diagram showing the suspension state of an alginate beide containing HepG2 cells when cultured in a culture medium.
[Fig. 11] is a diagram showing the suspension state of collagen gel capsules in which HepG2 cells are embedded, when they are cultured in a culture medium.
[Fig. 12] is a diagram showing the suspension state when the rice-derived union tissue is cultured in the medium composition.
[Fig. 13] shows the viscosity of each medium composition at 25 캜.
14 is a scanning electron micrograph of the MNC-containing medium composition of Example 1. Fig.
15 is a scanning electron micrograph of the PNC-containing medium composition of Example 2. Fig.
Fig. 16 shows a scanning electron microscope photograph of the CT-containing medium composition of Example 3. Fig.
17 is a scanning electron micrograph of the DAG-containing medium composition of Example 4. Fig.
18 is a scanning electron micrograph of the Car-containing medium composition of Example 5. Fig. Dry at room temperature.
19 is a scanning electron micrograph of the Car-containing medium composition of Example 5. Fig. Dry at 110 ° C.
Fig. 20 is a scanning electron microscope photograph of the Xan-containing medium composition of Comparative Example 3. Fig.
21 is a scanning electron micrograph of the DU-containing medium composition of Comparative Example 4. Fig.
22 shows the result of observation of the dispersion state of HepG2 cell sphere after suspending the sperm of HepG2 cells for 6 days in the MNC-containing medium composition of Example 1. Fig. From the left, the MNC concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%.
23 shows the result of observation of the dispersed state of HepG2 cell sphere after suspension culture of HepG2 cells for 6 days in the PNC-containing medium composition of Example 2. Fig. From the left, the PNC concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%.
24 shows the result of observation of the dispersion state of HepG2 cell sphere after suspending the sphere of HepG2 cells for 6 days in the CT-containing medium composition of Example 3. Fig. From the left, the CT concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%.
25 shows the result of observation of the dispersion state of HepG2 cell sphere after suspending the sphere of HepG2 cells for 6 days in the DAG-containing medium composition of Example 4. Fig. From the left, the DAG concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%.
[Fig. 26] shows the result of observation of the dispersion state of HepG2 cell sphere after suspending the sphere of HepG2 cells for 6 days in the Car-containing medium composition of Example 5. Fig. From the left, the Car concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%.
[Fig. 27] shows observation results of the state of dispersion of HepG2 cell sphere after suspension culture of HepG2 cells for 6 days in the Xan-containing medium composition of Example 5. Fig. From the left, the Xan concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%.
28 shows the result of observation of the dispersion state of HepG2 cell sphere after suspension culture of HepG2 cells for 6 days in the DU-containing medium composition of Comparative Example 4. Fig. From the left, the DU concentrations are 0.01, 0.03, 0.05, 0.07 and 0.1 w / v%.
FIG. 29 shows the result of observation of the dispersion state of HepG2 cell sphere after suspension culture of HepG2 cells in an Alg-containing medium composition of Comparative Example 5 for 6 days. From the left, Alg concentrations are 0.01, 0.03, 0.05, 0.07, and 0.1 w / v%.
[Fig. 30] shows the RLU value at 6 days after initiation of suspension culture of MCF7 cells in the medium composition of Example 1 'and Comparative Example 5'.
[Fig. 31] shows the RLU value at 6 days after initiation of suspension culture of MCF7 cells in the medium compositions of Examples 2 'and 3'.
[Fig. 32] shows the RLU value at 6 days after initiating suspension culture of MCF7 cells in the medium composition of Example 4 'and Example 5'.
[Fig. 33] shows the RLU value at 6 days after initiation of suspension culture of MCF7 cells in the medium composition of Comparative Examples 3 'and 4'.
[Figure 34] shows the RLU value at 6 days after initiation of suspension culture of A375 cells in the medium composition of Example 1 'and Comparative Example 5'.
[Figure 35] shows the RLU value at 6 days after initiation of suspension culture of A375 cells in the medium compositions of Examples 2 'and 3'.
[Figure 36] shows the RLU value at 6 days after initiation of suspension culture of A375 cells in the medium composition of Example 4 'and Example 5'.
[Figure 37] shows the RLU value at 6 days after initiation of suspension culture of A375 cells in the medium composition of Comparative Examples 3 'and 4'.
[Fig. 38] is a result of microscopic observation of the dispersion state of MCF7 cells in each culture medium on the second day of suspension culture.
FIG. 39 is a microscopic observation of the state of dispersion of A375 cells in each culture medium on the second day of suspension culture.
[Fig. 40] is a result of microscopic observation of the dispersion state of MDCK cells in each culture medium on the 4th day of suspension culture initiation.

Hereinafter, the present invention will be described in more detail.

The terms used in this specification are defined as follows.

In the present invention, a cell is the most basic unit constituting an animal and a plant, and has a cytoplasm and various organ organelles inside the cell membrane as its element. In this case, the nucleus that encapsulates the DNA may or may not be contained within the cell. For example, in the present invention, the cells derived from an animal include, but are not limited to, gonads such as sperm, oocytes, somatic cells constituting a living body, stem cells, progenitor cells, cancer cells isolated from a living body, (cell line) that acquires immortalizing ability and remains stably in vitro, a cell that is separated from a living body and artificially genetically modified, a cell that is separated from a living body (where the nucleus is artificially exchanged), and the like. Examples of somatic cells constituting the living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, mononuclear leukocytes, bone cells, bone marrow cells, A cell, a keratinocyte, an adipocyte, a mesenchymal cell, an epithelial cell, a epidermal cell, an endothelial cell, a vascular endothelial cell, a liver parenchymal cell, a cartilage cell, a cumulus cell, a neuron cell, a glial cell, a neuron, (Eg, smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells, and mononuclear cells, and the like. The somatic cells may be, for example, skin, kidney, spleen, adrenal gland, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, spleen, bladder, prostate, testis, thymus, muscle, connective tissue, , Blood vessels, blood, heart, eyes, brain, neural tissue, and the like. Stem cells are cells that have both the ability to replicate themselves and the ability to differentiate into other multiple lines. Examples include, but are not limited to, embryonic stem cells (ES cells), embryonal tumor cells, embryonic stem cells, artificial pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, , Liver stem cells, pancreatic stem cells, muscle stem cells, reproductive stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells and the like. A progenitor cell is a cell that is on the way to differentiate into somatic cells or germ cells from the above-mentioned stem cells. Cancer cells are cells derived from somatic cells and having acquired infinite proliferation ability. The cell line is a cell that has obtained infinite proliferative capacity by an artificial manipulation in vitro. Examples include, but are not limited to, CHO (Chinese hamster ovary cell line), HCT116, Huh7, HEK293 (human fetal kidney cell) MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3 (human leukemia cell line), HeLa (human cervical cancer cell line), HepG2 , PC12, S2, Sf9, Sf21, High Five (registered trademark), Vero, and the like.

The plant-derived cells in the present invention include cells separated from each tissue of the plant and protoplasts obtained by artificially removing cell walls from the cells.

The tissue in the present invention is a unit of a structure in which cells having some types of different characteristics and functions are collected in a specific manner. Examples of animal tissues include epithelial tissues, connective tissues, muscle tissues, nerve tissues and the like. Examples of the tissue of the plant include fissured tissue, epidermal tissue, anatomical tissue, follicular tissue, ductal tissue, mechanical tissue, soft tissue, demineralized cell mass (unified tissue: callus), and the like.

When cells and / or tissues are cultured, the cells and / or tissues to be cultured can be freely selected and cultured from the cells and / or tissues described above. Cells and / or tissues may be recovered directly from the animal or plant. Cells and / or tissues can be derived, grown, transformed, and then harvested from an animal or plant by applying a specific treatment. In this case, the treatment may occur in vivo or ex vivo. Examples of animals include fish, amphibians, reptiles, birds, crustaceans, hexagons, mammals, and the like. Examples of mammals include but are not limited to rats, mice, rabbits, guinea pigs, squirrels, hamsters, rodents, platypuses, dolphins, whales, dogs, cats, goats, cows, horses, sheep, Pike, squirrel monkey, squirrel monkey, rhesus monkey, chimpanzee and human. The plant is not particularly limited as long as the collected cells and / or tissues can be liquid cultured. Examples thereof include plants (for example, ginseng, bing cats, alopecia, chrysanthemum, belladonna, etc.), cosmetics or food raw materials which produce vital drugs (for example, saponins, alkaloids, berberine, scopolin, plant sterols, (E.g., blueberry, safflower, madderney, caprolactone, etc.) producing a colorant or polysaccharide (for example, anthocyanin, safflower dye, daffodil dye, Plants, and the like.

The suspension of cells and / or tissues in the present invention refers to a state in which cells and / or tissues are not attached (non-attached) to a culture container. In addition, in the present invention, when cells and / or tissues are to be proliferated, differentiated or maintained, the cells and / or tissues may be exposed to the liquid medium composition from the outside under pressure or vibrations thereof or in the absence of agitation, The state of uniformly dispersed and suspended in the liquid medium composition is represented by "suspension ", and the culture of cells and / or tissue under the above conditions is represented by" suspension culture. " In the "suspension state", the suspension period is at least 5 minutes, preferably at least 1 hour, at least 24 hours, at least 48 hours, at least 6 days, at least 21 days, but as long as the suspension is maintained, It does not.

In a preferred embodiment, the culture medium composition of the present invention is capable of suspending cells and / or tissues at least at a point within a temperature range (for example, 0 to 40 ° C) in which cells and tissues can be maintained or cultured Do. The medium composition of the present invention is capable of suspending cells and / or tissues at at least one point within a temperature range of preferably 25 to 37 占 폚, most preferably 37 占 폚.

For example, polystyrene beads (Size 500-600 占 퐉, manufactured by Polysciences Inc.) are uniformly dispersed in the medium composition of the evaluation target, allowed to stand at 25 占 폚 for at least 5 minutes (Preferably, 24 hours or more, 48 hours or more) and observing whether the suspended state of the cell can be maintained.

The culture medium composition of the present invention is a composition containing a nanofiber and a culture medium capable of culturing cells or tissues in a suspended state (preferably capable of suspension culture).

The medium composition is preferably a composition which permits the exchange of the medium composition during the culture and the recovery of the cells or tissues after completion of the culture. More preferably, it is a composition that does not require any of temperature change, chemical treatment, enzyme treatment and shearing force for cell or tissue recovery.

[Nano Fiber]

The nanofibers contained in the medium composition of the present invention exhibit an effect of uniformly suspending cells and / or tissues in a liquid medium. More specifically, nanofibers formed in a liquid medium by a covalent bond or a self-organizing low-molecular weight compound or polymer compound through ionic bonding, electrostatic interaction, hydrophobic interaction, van der Waals force, Or the like can be referred to as nanofibers contained in the medium composition of the present invention. In the medium composition of the present invention, nanofibers form a three-dimensional network, which is not constrained to the theory, but which supports cells and tissues, thereby keeping the cells and tissues suspended.

In this specification, the nanofiber refers to a fiber having an average fiber diameter (D) of 0.001 to 1.00 μm. The average fiber diameter of the nanofiber used in the present invention is preferably 0.005 to 0.50 mu m, more preferably 0.01 to 0.05 mu m, and still more preferably 0.01 to 0.02 mu m. If the average fiber diameter is less than 0.001 탆, the nanofiber may become too fine to prevent the suspension effect from being achieved, and the properties of the medium composition containing it may not be improved.

The aspect ratio (L / D) of the nanofiber used in the present invention is usually 2 to 500, preferably 5 to 300, and more preferably 10 to 250, which is obtained from the average fiber length / average fiber diameter. When the aspect ratio is less than 2, the dispersibility in the medium composition may be insufficient, and there is a possibility that a sufficient suspension effect may not be obtained. When the ratio is more than 500, the fiber length becomes very large. In this case, by raising the viscosity of the composition, it means that there is a possibility that the operation for transferring such as the medium exchange or the like may be hindered. Further, since the culture medium is difficult to transmit visible light, the transparency is lowered, and the time-course observation of the cultured cells becomes difficult, and there is a possibility that evaluation of cells using absorption, fluorescence, luminescence, etc. may be hindered.

In the present specification, the average fiber diameter (D) of the nanofibers was measured as follows. First, Okenshoji Co., Ltd. Lt; RTI ID = 0.0 > JEOL < / RTI > Hydrophilic treatment was performed for 3 minutes with a commercially available ion creaner (JIC-410), and several droplets of the nanofiber dispersion of the evaluation target (diluted with ultrapure water) were added dropwise and dried at room temperature. Hitachi, Ltd. (TEM, H-8000) (10,000 times) with an accelerating voltage of 200 kV. Using the obtained images, one fiber diameter was measured for each of the nanofibers (number of specimens: 200 to 250), and the average value thereof was defined as the average fiber diameter (D).

The average fiber length (L) was measured as follows. The nanofiber dispersion as an evaluation target was diluted with purified water to 100 ppm, and the nanofibers were uniformly dispersed using an ultrasonic washing machine. This nanofiber dispersion liquid was cast on a silicon wafer which had been previously subjected to hydrophilization treatment using concentrated sulfuric acid and dried at 110 DEG C for 1 hour to be used as a sample. One fiber length was measured for each nanofiber (sample number: 150 to 250) using the image obtained by observing the obtained sample with a scanning electron microscope (SEM, JSM-7400 F) (2,000 times) The average value was taken as the average fiber length (L).

The nanofibers to be used in the present invention can be uniformly dispersed into the liquid while maintaining the primary fiber diameter while mixing with the liquid medium so that the nanofibers are uniformly dispersed into the liquid and the cells and / And has the effect of preventing the sedimentation. By "not substantially increasing the viscosity of the liquid" means that the viscosity of the liquid does not exceed 8 mPa · s. The viscosity of the liquid at this time (that is, the viscosity of the medium composition produced by the production method of the present invention) is 8 mPa · s or less, preferably 4 mPa · s or less, more preferably 2 mPa · s or less. In addition, when the nanofibers are dispersed in the liquid medium and the cells and / or tissues are uniformly suspended (preferably suspended) while the viscosity of the liquid is not substantially increased, Chemical structure, molecular weight, physical properties, etc. are not limited.

The viscosity of the liquid containing the nanofibers can be measured, for example, by the method described in the following Examples. Specifically, it can be evaluated using a sound bending vibration viscometer (SV-1A, A & D Company Ltd.) under the condition of 25 ° C.

Examples of raw materials constituting the nanofibers include, but are not limited to, low molecular weight compounds and polymer compounds.

Preferred examples of the low-molecular weight compound used in the present invention are not particularly limited, and examples thereof include amino acid derivatives such as L-isoleucine derivatives, L-valine derivatives and L-lysine derivatives, trans- Cyclohexanediamine derivatives such as cyclohexanedimethanolamine derivatives, cyclohexanediamine derivatives such as cyclohexanedicarboxylic acid diamide derivatives, cyclohexanediamine derivatives such as cyclohexanedimethanolamine derivatives, Low-molecular gels such as cyclohexanetricarboxamide, 2,4-dibenzylidene-D-sorbitol, N-lauroyl-L-glutamic acid- ?,? -Bis-n-butylamide and calcium dehydroabietate Topic.

Preferable specific examples of the polymer compound used in the present invention are not particularly limited, and examples thereof include polysaccharides and polypeptides.

Polysaccharide means a glycopolymer in which 10 or more monosaccharides (e.g., triose, tetrose, pentose, hexose, heptose, etc.) are polymerized. Polysaccharides include non-water-soluble polysaccharides and water-soluble polysaccharides.

Examples of the water-insoluble polysaccharide include celluloses such as cellulose and hemicellulose; And chitin components such as chitin and chitosan, but are not limited thereto.

Examples of the water-soluble polysaccharide include acidic polysaccharides having an anionic functional group. Examples of the acidic polysaccharide having an anionic functional group include, but are not limited to, polysaccharides having uronic acids (for example, glucuronic acid, diuronic acid, galacturonic acid, mannuronic acid) in the structure; Polysaccharides having a sulfuric acid or phosphoric acid in the structure, and polysaccharides having both structures. More specifically, there may be mentioned hyaluronic acid, gellan gum, deacylated gellan gum (DAG), rhamzaen gum, dianthan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, Polymeric compounds composed of at least one kind selected from the group consisting of phosphate, heparin, heparin sulfate, keratosulfate, chondroitin sulfate, deramatan sulfate, rhamnsulfate, alginic acid and salts thereof are exemplified.

Examples of the salt include alkali metal salts such as lithium, sodium and potassium; Alkaline earth metal salts such as calcium, barium and magnesium; Salts of aluminum, zinc, copper, iron and the like; Ammonium salts; Quaternary ammonium salts such as tetraethylammonium, tetrabutylammonium, methyltributylammonium, cetyltrimethylammonium, benzylmethylhexyldecylammonium and choline; Salts with organic amines such as pyridine, triethylamine, diisopropylamine, ethanolamine, diolamine, tromethamine, meglumine, procaine, chloropropane and the like; And salts with amino acids such as glycine, alanine, valine and the like.

As the polypeptide, there may be mentioned a polypeptide constituting a fiber in a living organism. Specific examples include, but are not limited to, collagen, elastin, myosin, keratin, amyloid, fibroin, actin, tubulin and the like.

The raw materials constituting the nanofibers used in the present invention include materials derived from microorganisms, genetically-engineered materials, and artificially synthesized materials using enzymes and chemical reactions, as well as materials derived from natural sources do. The raw material constituting the nanofibers used in the present invention is preferably a substance derived from a naturally occurring substance (that is, a naturally extracted substance) or a substance obtained by modifying it by a chemical reaction or an enzymatic reaction.

In one embodiment, the polysaccharide is a water-insoluble polysaccharide. Preferred non-aqueous polysaccharides include cellulose; And chitin components such as chitin and chitosan. Cellulose and chitin are most preferred in view of the low viscosity of the culture medium composition and the ease of recovery of cells or tissue.

Cellulose is a natural polymer compound in which D-glucopyranose, which is a six-atom loop of glucose, is? -1,4 glucoside-bonded. Examples of raw materials include plant-derived celluloses such as wood, bamboo, hemp, jute, kenaf, cotton, crops and food residues; or bacterial cellulose, Cladophora, Glaucocystis, Valonia, Tunicate cellulose and the like can be used. Plant-derived cellulose is a bundle of very thin fibers called microfibrils that form a stepwise higher order structure such as fibrils, lamellar and fibrous cells. Also, in bacterial cellulose, cellulose microfibrils, which are secreted from bacterial cells and have a constant thickness, form a fine net structure.

In the present invention, high purity cellulose raw materials such as cotton, bacterial cellulose and the like can be directly used. However, other plant-derived cellulose and the like are preferably used after isolation and purification. Examples of the cellulose that is preferably used in the present invention include cotton cellulosic, bacterial cellulose, kraft pulp cellulose, microcrystalline cellulose and the like. Particularly, kraft pulp cellulose is particularly preferably used since it has a high suspending action.

The chitin component means one or more carbohydrates selected from the group consisting of chitin and chitosan. The major sugar units that make up chitin and chitosan are N-acetylglucosamine and glucosamine, respectively. Generally, chitin has a high content of N-acetylglucosamine and is insoluble in acidic aqueous solutions, chitosan has a high content of glucosamine and is soluble in acidic aqueous solutions. In the present specification, for convenience, the ratio of N-acetylglucosamine to constitutional sugar is 50% or more and chitin is less than 50% is called chitosan. From the viewpoint of attaining a high suspending action, it is more preferable that the ratio of N-acetylglucosamine is high among sugar units constituting chitin. The ratio of N-acetylglucosamine in sugar units constituting chitin is preferably 80% or more, more preferably 90% or more, still more preferably 98% or more, and most preferably 100%.

As a raw material of chitin, for example, many living resources such as shrimp, crab, insect, shellfish, mushroom and the like can be used. The chitin used in the present invention may be chitin having an a-type crystal structure such as crab shell and shrimp shell derived chitin or may be chitin having a crystal structure of a beta -form such as chitin derived from bone of squid It is possible. The outer shells of crab and shrimp are often regarded as industrial wastes, and are preferred as raw materials from the standpoint that they are readily available and effectively used. On the other hand, a protein removal process and a demineralization process are required to remove proteins and ashes contained as impurities. Therefore, in the present invention, it is preferable to use purified chitin already subjected to matrix elimination treatment. Purified chitin is commercially available.

In one embodiment, the polysaccharide is a water soluble polysaccharide. Preferred water-soluble polysaccharides include deacylated gellan gum, carrageenan, and the like. From the viewpoint of attaining a high suspension action, deacylated gellan gum is most preferable.

Deacylated gellan gum is a linear polymeric polysaccharide containing as its constituent units four molecules of glucose, 1-3 bonded glucose, 1-4 bonded glucuronic acid, 1-4 bonded glucose and 1-4 bound rhamnose, In the formula (I), R1 and R2 are each a hydrogen atom and n is a polysaccharide having an integer of 2 or more. Provided that R 1 may contain a glyceryl group, R 2 may contain an acetyl group, and the content of the acetyl group and the glyceryl group is preferably 10% or less, more preferably 1% or less.

As a method of producing gellan gum, a production microorganism is cultivated in a fermentation medium, and mucosal components produced outside the microorganism are recovered by a conventional purification method, and then pulverized after drying, pulverization and the like. In addition, deacylated gellan black is obtained by culturing a microorganism producing gellan gum in a fermentation medium and recovering mucosal secretion produced from the outside of the microorganism. In this case, when the mucous membrane component is recovered, an alkali treatment may be performed to recover the glyceryl group and acetyl group bound to the 1-3 bonded glucose moieties after deacylation. Purification of the deacylated gellan gum from the recovered mucosal secretion can be carried out, for example, by liquid-liquid extraction, fractional precipitation, crystallization, various ion exchange chromatography, gel filtration chromatography using Sephadex LH-20 or the like, Adsorption chromatography with silica gel or the like, adsorption / desorption treatment of active material by thin layer chromatography, high performance liquid chromatography using a reversed phase column, and the like, and these may be carried out singly or in any combination or repeatedly . Examples of the producing microorganism of gellan gum include, but are not limited to, microorganisms obtained by modifying the genes of Sphingomonas elodea and Sphingomonas elodea.

In the case of the deacylated gellan gum, commercially available ones, for example, SANSHO Co., Ltd. &Quot; KELCOGEL (registered trademark of CP Kelco) CG-LA ", San-Ei Gen F.F.I., Inc. &Quot; KELCOGEL (registered trademark of CP Kelco) ", etc. may be used.

The weight average molecular weight of the polymer compound used in the present invention is preferably 1,000 to 50,000,000, more preferably 10,000 to 20,000,000, and still more preferably 100,000 to 10,000,000. For example, the molecular weight can be inferred from gel permeation chromatography (GPC) based on polyethylene glycol, conversion to pullulan, and viscosity of an aqueous solution.

In the present invention, a plurality of (preferably two) polymer compounds mentioned above may be used in combination. The kind of combination of the polymer compound is not particularly limited so long as the nanofiber is formed in the liquid medium or dispersed as the nanofiber to suspend the cells and / or tissues without increasing the viscosity of the liquid medium (preferably, And is not particularly limited. Preferably, the combination comprises at least cellulose, chitin, collagen, or deacylated gellan gum. That is, preferred combinations of polymer compounds include cellulose, chitin, collagen, or deacylated gellan gum; And other polymer compounds (for example, xanthan gum, alginic acid, carrageenan, diugane gum, methyl cellulose, locust bean gum or salts thereof).

[Preparation of nanofiber]

The culture medium composition of the present invention contains nanofibers prepared from the above-described raw materials. The method of preparing nanofibers is a method in which a water-insoluble polymer compound (for example, a non-water-soluble polysaccharide such as cellulose or chitin) is used as a raw material and a water-soluble polymer compound (for example, a water-soluble polysaccharide such as deacylated gellan gum) . ≪ / RTI >

When the raw material of the nanofiber is a water-insoluble polymer compound (for example, a water-insoluble polysaccharide such as cellulose or chitin), the nanofiber is generally obtained by pulverizing the raw material. The grinding method is not limited. However, in order to make the fiber diameter and fiber length to be mentioned below satisfying the object of the present invention, it is preferable to use a grinding mill such as a medium agitating mill such as a high pressure homogenizer, a grinder (millstone), a bead mill, A method of providing a strong shearing force is preferable.

Among them, it is preferable to make fine using a high-pressure homogenizer, and it is preferable to make it fine (pulverized) by using a wet grinding method as disclosed in, for example, Japanese Patent Application Laid-Open No. 2005-270891 or Japanese Patent No. 5232976 . Specifically, the raw material is pulverized by spraying the dispersion of raw materials at a high pressure from a pair of nozzles and colliding with each other, and the raw material is pulverized, for example, by Star Burstsystem (high pressure grinding apparatus manufactured by Sugino Machine Limited) or NanoVater (yoshida kikai co. , a high-pressure grinding apparatus manufactured by ltd) are used for this.

The degree of micronization and homogenization when the raw material is micronized (pulverized) by using the above-described high-pressure homogenizer is determined by the pressure to be pumped into the ultra-high pressure chamber of the high-pressure homogenizer, the number of times (the number of processing) And the concentration of the raw material in the aqueous dispersion. The pump pressure (process pressure) is generally 50 to 250 MPa, preferably 150 to 245 MPa. When the pump pressure is less than 50 MPa, the nanofibers become insufficient in fineness, and the effect expected by the miniaturization may not be achieved.

The concentration of the raw material in the aqueous dispersion during the refinement treatment is 0.1% by mass to 30% by mass, preferably 1% by mass to 10% by mass. When the concentration of the raw material in the aqueous dispersion is less than 0.1% by mass, the productivity is lowered. When the concentration is higher than 30% by mass, the pulverization efficiency is lowered, and desired nanofibers can not be obtained. The number of times of fineness (pulverization) is not particularly limited, but depends on the concentration of the raw material in the aqueous dispersion. When the concentration of the raw material is 0.1 to 1 mass%, it is sufficiently fine by 10 to 100 times of processing, but about 1 to 10 mass% requires about 10 to 1000 times of processing. In the case of a high concentration exceeding 30% by mass, the number of times of treatment is required several thousands of times, but the viscosity is too high to perform normal handling, and therefore, it is industrially unrealistic.

When the raw material of the nanofiber includes a water-soluble polymer compound (for example, a water-soluble polysaccharide such as a deacylated gellan gum), when the substance is added to a medium, the substance is collected through metal cations in the medium, Nanofibers are formed, and this forms a three-dimensional network. As a result, nanofibers are formed that can culture the cells or tissues in a suspended state.

The concentration of the nanofibers in the culture medium composition of the present invention can be suitably determined so as to suspend the cells and / or tissues without substantially increasing the viscosity of the culture medium (preferably suspending the cells and / or tissues). (Weight / volume), for example from 0.0005% to 1.0% (weight / volume), preferably from 0.001% to 0.5% (weight / volume), more preferably from 0.005% to 0.1 % (Weight / volume), more preferably 0.005% to 0.05% (weight / volume).

For example, in the case of cellulose nanofibers, it is generally 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% , More preferably 0.01% to 0.1% (weight / volume), and more preferably 0.01% to 0.05% (weight / volume).

In the case of the pulp cellulose nanofibers among the cellulose nanofibers, the lower limit of the concentration in the medium is preferably 0.01% (weight / volume) or more, 0.015% or less, (Weight / volume) or more, 0.02% (weight / volume) or more, 0.025% (weight / volume) or more, or 0.03% (weight / volume) or more. In the case of pulp cellulose nanofibers, the upper limit of the concentration in the medium is preferably not more than 0.1% (weight / volume) or not more than 0.04% (weight / volume) from the viewpoint of not substantially increasing the viscosity of the medium .

In the case of the microcrystalline cellulose nanofiber, the lower limit of the concentration in the medium is preferably 0.01% (weight / volume) or more, 0.03% (weight / volume) or more, or 0.05% Or more. From the viewpoint of enabling suspension culture, the lower limit of the concentration of microcrystalline cellulose nanofibers in the culture medium is preferably 0.03% (weight / volume) or more, or 0.05% (weight / volume) or more. In the case of microcrystalline cellulose nanofibers, the upper limit of the concentration in the medium is preferably 0.1% (weight / volume) or less.

In the case of chitin nanofibers, it is usually 0.0001% to 1.0% (weight / volume), for example 0.0005% to 1.0% (weight / volume), preferably 0.001% to 0.5% (weight / volume) Is added in the medium at 0.01% to 0.1% (weight / volume), most preferably 0.03% to 0.07% (weight / volume). From the viewpoint of the expression of the suspension action, the lower limit of the concentration of chitin nanofiber in the culture medium is preferably 0.0001% (weight / volume) or more, 0.0003% (weight / volume) (Weight / volume). From the viewpoint of enabling the suspension culture, the lower limit of the chitin nanofiber in the culture medium is preferably 0.03% (weight / volume) or more. The upper limit of the concentration of chitin nanofiber in the culture medium is preferably 0.1% (weight / volume) or less.

For water-insoluble nanofibers such as cellulose nanofibers and chitin nanofibers, if the concentration is usually 0.1% (weight / volume) or less, the viscosity of the culture medium composition is not substantially increased.

(Weight / volume), preferably 0.001% to 0.5% (weight / volume), more preferably 0.01% to 0.1% (weight / volume) and most preferably 0.02% % To 0.1% (weight / volume). The lower limit of the concentration of carrageenan in the medium is preferably 0.01% or more. The upper limit of the concentration of carrageenan in the medium is preferably 0.1% (weight / volume) or less from the viewpoint of the expression of the suspension action and from the viewpoint of enabling suspension culture. From the viewpoint of not substantially increasing the viscosity of the medium, it is also preferable to set the upper limit of the carrageenan to 0.04% (weight / volume) or less.

In the case of the deacylated gellan gum, it is usually 0.001% to 1.0% (weight / volume), for example 0.005% to 1.0% (weight / volume), preferably 0.003% to 0.5% (Weight / volume), more preferably 0.01% to 0.05% (weight / volume), and most preferably 0.01% to 0.02% (weight / volume). From the viewpoint of the expression of the suspension action, the lower limit of the concentration of the desaturated gellan gum in the medium is preferably 0.005% (weight / volume) or more, or 0.01% or more. From the viewpoint of enabling the suspension culture, the lower limit of the concentration of the desaturated gellan gum in the medium is preferably 0.01% (weight / volume) or more. From the viewpoint of not substantially increasing the viscosity of the medium, the upper limit of the concentration of the desaturated gellan gum in the medium is 0.05% (weight / volume) or less. From the viewpoint of not substantially increasing the viscosity of the culture medium, it is also preferable that the upper limit of the amount of the deacylated gellan gum in the culture medium is 0.04 (weight / volume)% or less.

[Combination of polysaccharides]

In addition to the nanofibers, a plurality of (preferably two) polysaccharides may be used in combination. The concentration of the polysaccharide can be suitably set as long as the cells and / or tissues are uniformly suspended (preferably in a suspended state) without substantially increasing the viscosity of the liquid medium. For example, when a combination of a nanofiber and a polysaccharide is used, the concentration of the nanofiber is 0.005 to 0.1% (weight / volume), preferably 0.01 to 0.07% (weight / volume) Is 0.005 to 0.4% (weight / volume), preferably 0.1 to 0.4% (weight / volume). Specific combinations of concentration ranges include the following.

Cellulose or chitin nanofibers: 0.005 to 0.1% (preferably 0.01 to 0.07%) (weight / volume)

Polysaccharide

Zanthan gum: 0.1 to 0.4% (weight / volume)

Sodium alginate: 0.1 to 0.4% (weight / volume) (preferably 0.0001 to 0.4% (weight / volume))

Locust bean gum: 0.1 to 0.4% (weight / volume)

Methyl cellulose: 0.1 to 0.4% (weight / volume) (preferably 0.2 to 0.4% (weight / volume))

Carrageenan: 0.05 to 0.1% (weight / volume)

Dianthum gum: 0.05 to 0.1% (weight / volume)

Native gellan gum: 0.0001-0.4% (weight / volume)

The concentration can be calculated from the following equation.

Concentration (%) = weight of nanofiber (g) / volume (ml) of media composition x 100

[Metal cation]

In one embodiment, the media composition of the present invention contains metal cations, such as divalent metal cations (such as calcium ion, magnesium ion, zinc ion, iron ion, and copper ion), preferably calcium ions. Particularly, when the nanofiber contained in the medium composition of the present invention is composed of a water-soluble polymer compound (for example, water-soluble polysaccharide such as deacylated gellan gum), the medium composition of the present invention contains the metal cation desirable. When a metal cation is contained, a collection of the water-soluble polymer compound (for example, a water-soluble polysaccharide such as a deacylated gellan gum) is formed through metal cations to form a nanofiber in the medium composition, thereby establishing a three-dimensional network. As a result, nanofibers capable of culturing cells or tissues in a suspended state can be formed.

[badge]

The medium contained in the medium composition of the present invention is Dulbecco's Modified Eagles's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum medium (EMEM), alphaMEM medium, Minimum Essential Medium, RPMI1640 medium, Iscove's Modified Dulbecco's Medium (IMDM) medium, MCDB131 medium, William (Manufactured by Cambrex Corporation), HPGM (manufactured by Cambrex Corporation), StemSpan H3000 (manufactured by Mitsubishi Chemical Corporation), StemPropan H3000 (manufactured by Mitsubishi Chemical Corporation), X-VIVO 15 (Invitrogen), mTeSR1 or 2 medium (manufactured by STEMCELL Technologies), Sf-900II (manufactured by Invitrogen Corporation), StemSpanSFEM (manufactured by STEMCELL Technologies), StemlineII (manufactured by Sigma Aldrich), QBSF-60 (Qualitybiological Co.), StemPro hESC SFM Article), Opti-Pro (Invitrogen Corp.), and the like.

When the cells and / or tissues are derived from plants, the cells are cultured in a Murashige Skoog (MS) medium, a Linsmaier Skoog (LS) medium, a White medium, a Gamborg's B5 medium, a Nickel medium, a Hela medium, a Morel medium ) Or a plant growth regulating component such as auxin and, if necessary, a plant growth regulator (such as a plant hormone), or the like, at a concentration suitable for a medium for modifying the medium to an optimum concentration (for example, ammonia nitrogen is halved) ) Can be referred to as a medium. Such a medium may be further supplemented with casein degrading enzyme, corn steep liquor, vitamins and the like as needed. Examples of the auxin include 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid . The auxin may be added to the medium, for example, at a concentration of about 0.1 to about 10 ppm. Examples of cytokinins include, but are not limited to, kinetin, benzyladenine (BA), zeatin, and the like. The cytokinin may be added to the medium, for example, at a concentration of about 0.1 to about 10 ppm.

Those skilled in the art can freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acid, sugar and the like to the above-mentioned medium according to the purpose. In the case of culture of cells and / or tissues derived from animals, one skilled in the art can add other chemical components and biocomponents in combination according to the purpose. Examples of components added to the medium for animal-derived cells and / or tissues include but are not limited to: fetal serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, Cellulosic materials such as bovine serum albumin, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various growth factors, various extracellular matrix, Molecules and the like. Examples of cytokines to be added to the medium include IL-1, IL-2, IL-3, IL-4, Interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin- Interleukin-11 (IL-11), Interleukin-12 (IL-12), Interleukin-13 (IL-13), Interleukin-14 (IL- Interferon-beta (IFN-?), Interferon-y (IFN-?), Granulocyte colony stimulating factor (G-CSF), interferon- (M-CSF), granule-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), flk2 / flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M , Erythropoietin (EPO), thrombopoietin (TPO), and the like, but are not limited thereto.

Examples of hormones added to the medium include melatonin, seratonin, tyrosine, triiodothyronine, epinephrine, norepinephrine, dopamine, Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin , Antidiuretic hormone, atrial sodium diuretic peptide, calcitonin, cholecystokinin, corticotropin releasing hormone, erythropoietin, follicle stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin releasing hormone, growth hormone releasing hormone, It has been shown that the growth hormone levels of gonadotropin, human placental lactogen, growth hormone, inhivin, insulin, insulin growth factor, leptin, luteinizing hormone, melanocyte stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, These include tin, thyroid stimulating hormone, thyrotropin releasing hormone, cortisol, aldosterone, te Estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandin, leukotriene, prostacyclin, thromboxane, prolactin releasing hormone, report But are not limited to, rofyn, brain sodium diuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, reninin and enkephalin.

Examples of growth factors added to the medium include transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF- beta), macrophage inflammatory protein- 1 alpha (MIP- (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8 or 9 Growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine, (IGFBP), insulin-like growth factor binding protein-1 (IGFBP), insulin-like growth factor binding protein-1 (IGFBP) Pleiotrophin, and the like, but are not limited thereto.

Such cytokines and growth factors having amino acid sequences that have been artificially altered by gene recombination techniques can also be added. Examples include IL-6 / soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

Examples of various extracellular matrices and various cell adhesion molecules include collagen I to XIX, fibronectin, bitronectin, laminin-1 to 12, nitogen, tenacin, trombospondin, von Willebrand factor, Dexamolines, desmoglene, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D - lysine, poly-L-lysine, chitin, chitosan, sepharose, hyaluronic acid, alginate gel, various hydrogels,

Examples of antibiotics added to the medium include, but are not limited to, sulfonamides and formulations, penicillin, penethicillin, methicillin, oxacillin, clock factin, dicloxycin, flucloc factin, napsilin, ampicillin, penicillin, amoxicillin, Cyclosporin, and derivatives thereof, oxolinic acid, amifloxacin, tembuproxacin, cyclosporin, cyclosporin, cyclosporin, cyclosporin, cyclosalicin, carbenicillin, ticarcillin, piperacillin, azulocillin, meslocillin, , Nalidixic acid, pyromidic acid, ciprofloxacin, cynoxazin, norfloxacin, purpleoxacin, loracicin, oproxacin, enoxacin, piperidic acid, sulbactam, clavulanic acid, , sulphamphylline, adinosiline and sulbactam formaldehyde hydrate ester, tazobactam, azoteon, sulfazethine, isosulfate tin, isosulfanthin, N-carboxycin, m-carboxyphenyl, Methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate, methyl pyruvate and methyl pyruvate.

[Production method of the medium composition]

The above-mentioned nanofibers may be cultured in a concentration that allows the cells and / or tissues to be suspended (preferably suspended) uniformly without substantially increasing the viscosity of the liquid medium , The above-mentioned medium composition of the present invention can be prepared. The present invention also provides a method of producing such a medium composition of the present invention.

The nanofiber may be in the form of a liquid, such as a dispersion in a suitable physiological aqueous solvent, or a substrate or a single substrate, in the form of a solid, such as a powder, tablet, pill, capsule, or granule. Examples of the additives used in the formulation include preservatives such as p-oxybenzoic acid ester; Excipients such as lactose, glucose, sucrose and mannitol; Lubricants such as magnesium stearate and talc; Binders such as poly (vinyl alcohol), hydroxypropylcellulose, and gelatin; Surfactants such as fatty acid esters; And plasticizers such as glycerin. These additives are not limited to those mentioned above, and can be freely selected if available to those skilled in the art. The sterilization method is not particularly limited, and for example, radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, filter sterilization and the like can be mentioned.

In a preferred embodiment, the media composition of the present invention is prepared by mixing the above-mentioned nanofibers with a liquid medium in a physiological aqueous solvent. The dispersion may be sterilized (autoclave, gamma sterilized, etc.). Alternatively, a liquid medium (an aqueous solution as a medium) prepared by dissolving the dispersion and the powder medium in water may be mixed and used after sterilization. The dispersion and the liquid medium may be individually sterilized before mixing. Examples of the aqueous solvent include water and dimethylsulfoxide (DMSO), but are not limited thereto. Water is preferred as the aqueous solvent. The aqueous solvent may contain suitable buffers and salts. The above-mentioned dispersion of nanofibers is useful as a media additive for preparing the medium composition of the present invention. The present invention also provides such media additives.

The mixing ratio of the nanofibers is usually 1:99 to 99: 1, preferably 10:90 to 90:10, and more preferably 20:80 to 80:20 as a dispersion liquid liquid medium (aqueous solution as a medium) .

In the case where the nanofiber is composed of a water-soluble polymer compound (for example, a water-soluble polysaccharide such as a deacylated gellan gum), the water-soluble polymer compound (for example, deacylated A water-soluble polysaccharide such as gellan gum) and a culture medium to form a nanofiber in the culture medium to prepare a culture medium composition of the present invention. The shape of the polymer compound may be a liquid such as a solution or suspension obtained by dissolving it in a suitable solvent using a powder, a tablet, a pill, a capsule, a granule, or a solubilizing agent, or may be bonded to a substrate or a carrier . Examples of the additives used in the formulation include preservatives such as p-oxybenzoic acid ester; Excipients such as lactose, glucose, sucrose and mannitol; Lubricants such as magnesium stearate and talc; Binders such as poly (vinyl alcohol), hydroxypropylcellulose, and gelatin; Surfactants such as fatty acid esters; And plasticizers such as glycerol. These additives are not limited to those mentioned above, and can be freely selected if they are available to those skilled in the art.

The above-mentioned polymer compounds can be sterilized as needed. The sterilization method is not particularly limited, and for example, radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization, filter sterilization and the like can be mentioned.

In a preferred embodiment, an aqueous solution of a water soluble polymeric compound (e.g., a water soluble polysaccharide such as a deacylated gellan gum) (used as Media Additive 2) is used in the process of the present invention. The aqueous solution can be obtained by dissolving the water-soluble polymer compound in a physiological aqueous solvent as a solid (e.g., a powder). Examples of the aqueous solvent include water and dimethylsulfoxide (DMSO), but are not limited thereto. Water is preferred as the aqueous solvent.

The aqueous solvent may contain suitable buffers or salts. The aqueous solvent may or may not contain a divalent metal cation, while a divalent metal cation is not included in the preferred embodiment. When the aqueous solvent does not contain a divalent metal cation, a water-soluble polymer compound (for example, a water-soluble polysaccharide such as a deacylated gellan gum) in an aqueous solution may form a nanofiber structure capable of culturing cells or tissues in a suspended state It can not be easily formed and can be stably stored in a dissolved state in water.

It is also possible to add an additive to the above-mentioned medium additive to improve the effect of the nanofiber or lower the concentration when using the additive. Examples of such additives include one or more polysaccharides including guar gum, tamarind gum, alginic acid propylene glycol ester, locust bean gum, gum arabic, tara gum, tamarind gum, methyl cellulose and the like.

Hereinafter, the production method of the culture medium composition of the present invention will be exemplified, but the present invention is not limited thereto. The nanofibers are added to ion exchange water or ultrapure water. Then, the mixture is stirred at room temperature and sterilized (for example, autoclaved at 121 캜 for 20 minutes) until a uniform dispersion is formed as a whole. The nanofiber dispersion is added while agitating (for example, a homomixer or the like) to the proposed medium to be used for political culture, and the solution is uniformly mixed with the medium. The mixing method of the aqueous solution and the medium is not particularly limited, and examples thereof include manual mixing such as pipetting, mixing using a device such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer.

(Weight / volume), preferably 0.001% -1.0% (weight / volume), more preferably 0.01-0.6% (weight / volume), for example, when preparing a culture composition using cellulose nanofibers. Cellulose nanofibers are added to ion-exchanged water or ultrapure water so that the concentration is% (weight / volume). Then, the mixture is stirred at room temperature until the whole is uniform, and sterilized (for example, autoclaved at 121 캜 for 20 minutes) is carried out. While the liquid medium such as a DMEM medium is stirred with a homomixer or the like, the aqueous solution is added to the liquid medium so as to have a desired final concentration (for example, when the final concentration is 0.03%, 0.6% aqueous solution: Is 1:20), the mixture is mixed homogeneously. Alternatively, a liquid medium such as a DMEM medium is added to the present aqueous solution to a desired final concentration by a pipette (for example, in the case of a final concentration of 0.03%, a ratio of 0.6% aqueous solution: medium is 1:20 ) And mix homogeneously by pipetting. The mixing method of the present dispersion and the medium is not particularly limited, and examples thereof include manual mixing such as pipetting or mixing using a device such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer.

[Culture method]

The present invention also relates to a culture method for growing cells or tissues using the above-mentioned culture medium composition of the present invention, a method for recovering the obtained cells or tissues by, for example, filtration, centrifugation or magnetic separation, Lt; RTI ID = 0.0 > of < / RTI >

The nanofiber used in the present invention has an effect of suspending the cells and / or tissues in a liquid containing the nanofibers when the cells and / or tissues are cultured in vitro (preferably an effect of suspending the cells and / or tissues) . Due to the suspension effect, it is possible to further increase the amount of cells and / or tissue per given volume, as compared with monolayer culture. In addition, when the conventional suspension culture method involves rotation or shaking, a shearing force acts on cells and / or tissues, so that the proliferation rate and recovery rate of cells and / or tissues are lowered, or the function of cells is impaired . The use of the culture medium composition of the present invention containing nanofibers can uniformly disperse cells and / or tissues without performing shaking or the like, and allows the intended cells and / or tissues to be easily . ≪ / RTI > Further, when suspending cells and / or tissues in a culture medium containing a conventional gel substrate, observation and recovery of cells and / or tissues are often difficult and the function thereof is often impaired during recovery. However, by using the culture medium containing the nanofibers of the present invention, the cells and / or tissues can be applied to the suspension culture, and their functions can be observed and recovered without any damage. In addition, conventional media containing gel substrates often have high viscosity and can make it difficult to exchange media. However, since the medium composition containing the nanofibers of the present invention has a low viscosity, it is possible to easily change the medium using a pipette, a pump or the like.

Cells and / or tissues derived from humans cultured by the method of the present invention can be transplanted for therapeutic purposes to a patient having a disease or disorder. In this case, the therapeutic target disease, the kind of the disorder, the pretreatment method, and the cell transplantation method are appropriately selected by those skilled in the art. The engraftment of the transplanted cells in the recipient, the recovery from the disease or disorder, the presence or absence of side effects accompanying the transplantation, and the effect of the treatment are appropriately examined and judged by the general method in transplantation treatment.

Moreover, since the cells and / or tissues are efficiently proliferated, the culture medium composition of the present invention can be used as a reagent for research on cells. For example, when analyzing factors regulating the differentiation and proliferation of cells and tissues, it is necessary to analyze the number and type of cells and the changes of cell surface differentiation markers and expression genes when cells and target factors are coexisted and cultured do. In this case, using the culture medium composition of the present invention not only efficiently amplifies the number of assay target cells, but also efficiently recovers. The culture conditions, the culture apparatus, the kind of the culture medium, the kind of the nanofiber of the present invention, the content of the nanofiber, the kind of the additive, the content of the additive, the culture period, the culture temperature, Is suitably selected by those skilled in the art from the range described. Cells proliferated or appeared by cultivation can be observed using a standard microscope in the art. In this case, the cultured cells can be stained using a specific antibody. The expression gene changed due to a desired factor can be detected by Northern blotting, RT-PCR, or the like, by extracting RNA (ribonucleic acid) from the cultured cells. In addition, the cell surface differentiation marker is detected by ELISA and flow cytometry using a specific antibody, and the effect on the differentiation and proliferation by the objective factor can be observed.

Since the cells and / or tissues are efficiently proliferated using the culture medium composition of the present invention, the culture method of the present invention is excellent as a method of propagating cells and / or tissues or a method of promoting the proliferation of cells and / or tissues. When cells and / or tissues are cultured using the culture medium composition of the present invention, the cells and / or tissues are dispersed while expanding three-dimensionally without adhering to the culture container or only on the bottom surface of the culture container, . Particularly, when chitin nanofiber is used as the nanofiber, the cell adheres to the chitin nanofiber and proliferates strongly from there as a scaffold. As a result, the proliferated cells, cell masses (spear, etc.) and / or tissue are grafted onto the nanofibers. This proliferation promoting effect requires the presence of a sufficient concentration of nanofibers in the culture medium to suspend the cells and / or tissues (that is, to avoid adhesion to cells and tissue culture containers) That is, the ability of the cells and / or tissues to be uniformly dispersed and suspended in the liquid medium composition without accompanying external pressure, vibration, shaking, rotational manipulation, etc.) capability is not essential. For example, in the case of chitin nanofibers, a concentration of less than 0.03% (weight / volume) (e.g., a concentration of less than 0.03% (wt / vol)) which allows stable suspension culture, , 0.025% (weight / volume) or less, 0.02% (weight / volume) or less), the proliferation promoting effect is also provided. In the case of microcrystalline cellulose nanofibers, a concentration of less than 0.03% (weight / volume) (for example, 0.025% (weight / volume)) which allows stable suspension cultivation if the concentration is more than 0.01% (weight / Or less, and 0.02% (weight / volume) or less), the proliferation promoting effect is also provided. In the case of deacylated gellan gum, a concentration of less than 0.01% (weight / volume) (for example, 0.009% (weight / volume)) which allows a stable suspension culture is obtained if the concentration is 0.005% Or less, and 0.008% (weight / volume) or less), a proliferation promoting effect is also provided.

Among the nanofibers, in particular chitin nanofiber, the cell proliferation promoting effect is excellent.

In the culture method of the present invention, both non-adherent cells and adherent cells can be used. Adherent cells require a scaffold for growth and proliferation. Non-adherent cells do not require a scaffold for growth and proliferation. In the culturing method of the present invention, adherent cells are preferably used. When adherent cells are used in the method of the present invention, the adherent cells are dispersed three-dimensionally while spreading, attached to the nanofibers without attaching to the bottom surface of the culture container, Proliferate in sphere state. In particular, when the chitin nanofiber is used as the nanofiber, the cell adheres to the chitin nanofiber, and from there, it proliferates strongly as a scaffold. As a result, the proliferated cells, cell mass (spear and the like) and / or tissue are grafted onto the nanofibers like a cluster of grapes. Thus, suspension culture of adherent cells can be performed. As a result, the proliferation of adherent cells can be promoted more than when cultured in the state of being attached to the bottom surface of the culture container. In addition, adherent cells can be cultured with higher density than when cultured in the state of being attached to the bottom surface of the culture container.

In the culture method of the present invention, since the suspension culture of the adhering cells can be carried out, the adhering cells are suspended and cultured by the culture method of the present invention, Adherent cells can be transferred by simply adding the culture medium composition of the present invention to the culture after culturing, or by adding all or part of the cultured culture medium to the fresh medium composition of the present invention. The present invention also provides a method for subculturing such adherent cells. Therefore, by using the subculture method of the present invention, the adherent cells can be subcultured without removing the cells from the culture container. In addition, by using the subculturing method of the present invention, the culture scale of the adhering cells can be expanded without performing the peeling operation of the cells from the culture container. As the peeling operation of the cells from the culture container, treatment with a chelating agent (e.g., EDTA) and / or a protease (e.g., trypsin, collagenase) may be mentioned. The subculturing method of the present invention is a method of subculturing an adherent cell (for example, an adherent cell whose viability is deteriorated by a peeling operation, an adherent cell whose characteristics are easily changed by a peeling operation) Lt; / RTI > Examples of adherent cells highly susceptible to the detachment of cells from a culture vessel include human pluripotent stem cells; Human precursor cells; Primary cells prepared from tissues such as hepatocytes, kidney cells, cartilage cells, blood vessel cells and adipocytes; MDCK cells, HEK293 cells, and CHO cells, and the like, but the present invention is not limited thereto.

Using the culture medium composition of the present invention, adherent cells can be cultured at a high density, and cells and / or tissues can be efficiently proliferated. Therefore, the culturing method of the present invention is useful for the production of useful substances by in vitro cell culture. The useful substance (s) can be obtained by applying a cell producing a useful substance to a suspension culture in a medium composition of the present invention and isolating a useful substance from the culture. Examples of useful substances include antibodies, enzymes (such as urokinase), hormones (such as insulin), cytokines (interferons, interleukins, tumor necrosis factors, colony stimulating agents, growth factors and the like), vaccine antigens and other physiologically active substances , Peptides, etc.), but are not limited to these. Cells that produce useful substances include, but are not limited to, skin cells, cartilage cells, hepatocytes, pancreatic cells, non-transformed cells such as kidney cells, and genes encoding useful substances or genes involved in biosynthesis of useful substances Cells. The cell producing the useful substance may be an adherent cell or a non-adherent cell, preferably an adherent cell. Preferably, the cell producing the useful substance secretes the useful substance out of the cell. Specific examples of cells that produce a useful substance include HEK293, CHO-K1, BHK-21, MDCK, Vero, HepG2, MCF-7, etc., into which a gene encoding a useful substance or a gene involved in biosynthesis of a useful substance is introduced But are not limited to these. Cells used for the production of useful substances such as recombinant proteins are well known to those skilled in the art, and such cells may be used in the methods of the present invention. The culture scale can be determined by adding the fresh medium composition of the present invention to the culture after the suspension culture of the adherent cells by the above-mentioned subculture method of the present invention, without performing the peeling operation of the cells from the culture vessel, Or by adding all or part of the culture after the culture to the fresh medium composition of the present invention. In order to isolate the useful substance from the culture, it is necessary to remove the cells from the culture. Since the viscosity of the culture medium composition of the present invention is not substantially increased by the addition of the nanofibers and the cells are suspended in the culture medium, the cells can be removed by a convenient method such as centrifugation or filtration. The nanofibers in the culture medium composition can also be removed by a convenient method such as centrifugation or filtration. Methods for isolating useful substances from cultures are well known to those skilled in the art and include, for example, chromatography (e.g., ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reversed phase chromatography, Biochemical separation and purification of physiologically active substances can be applied.

When the cells and / or tissues are cultured using the culture method of the present invention, culturing tools generally used for culturing cells such as a chalet, a flask, a plastic pack, a Teflon (registered trademark) bag, a dish, a chalet, a tissue culture A microwave plate, a multi-plate, a multi-well plate, a chamber slide, a tube, a tray, a culture bag, a roller bottle and the like can be used for culture. The material of the culturing tool is not particularly limited, and examples thereof include glass, polyvinyl chloride, cellulose-based polymer, polystyrene, polymethylmethacrylate, polycarbonate, polysulfone, polyurethane, polyester, polyamide, polystyrene , Plastics such as polypropylene, and the like can be mentioned. In addition, a variety of surface treatments (e.g., plasma treatment, corona treatment, etc.) can be applied to these plastics. Furthermore, these culture tools can be coated with an extracellular matrix, a cell adhesion molecule, or the like in advance. Examples of such coating materials include collagen I to XIX, fibronectin, bitonectin, laminin-1 to 12, nitogen, tenacin, trombospondin, von Willebrand factor, osteopontin, fibrinogen, Various kinds of elastin, various proteoglycans, various cardinalin, desmocolin, desmoglene, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin, hyaluronic acid, superfamily, , Poly-L-lysine, chitin, chitosan, sepharose, alginic acid gel, hydrogel, and cleavage fragments thereof. These coating materials in which amino acid sequences are artificially modified by gene recombination techniques can also be used. It is also possible to use a coating material for inhibiting adhesion of cells and / or tissues to a culture tool. Examples of such a coating material include silicon, poly (2-hydroxymethyl methacrylate), poly (2-methacryloyloxyethylphosphoryl choline), and the like, but are not limited thereto.

Culturing of cells and / or tissues can be carried out under mechanical control by automatically executing cell seeding, medium exchange, cell image acquisition, and cultured cell collection under a closed environment, thereby controlling pH, temperature, oxygen concentration and the like at high density It may be carried out by a bioreactor capable of culturing or an automatic culture apparatus. Feed-batch culture, continuous culture and perfusion culture are available as methods for supplying new medium during cultivation using these devices and supplying the required substance to cells and / or tissues, Can be used.

Those skilled in the art can freely select the type and state of cells and / or tissues to be cultured by the method of the present invention. Specific preferred examples thereof include, but are not limited to, a state in which cells and / or tissues are dispersed in a medium composition alone, a state in which cells and / or tissues adhere to the surface of a carrier, cells and / A state where a plurality of cells aggregate and form cell aggregates (spears), or a state where two or more cells aggregate and form cell aggregates (sphere). More preferred is a state in which cells and / or tissues adhere on the surface of a carrier, a state in which cells and / or tissues are embedded in a carrier, a state in which a plurality of cells aggregate and form cell aggregates (sphere) Cells aggregate and form cell aggregates (sphere). More preferably, a state in which cells and / or tissues adhere on the surface of a carrier, a state in which a plurality of cells aggregate and form a cell aggregate (sphere), and a state in which two or more cells aggregate and form a cell aggregate (sphere) to be. In the state in which cell aggregates (sphere) are formed in such a state, cell-cell interaction and cell structure close to those of the in-vivo environment can be reconstructed, long-term culture can be performed while maintaining cell function, Can be referred to as the most preferable state to be cultured by the culture method of the present invention.

Examples of carriers for supporting cells and / or tissues on the surface include microcarriers composed of various polymers, glass beads, and ceramic beads. Examples of the polymer include a resin such as a vinyl resin, a urethane resin, an epoxy resin, polystyrene, polymethylmethacrylate polyester, polyamide, polyimide, silicone resin, phenol resin, melamine resin, urea resin, aniline resin, Cellulose, collagen, dextran, gelatin, cellulose, alginate, and mixtures thereof. The carrier may be coated with a compound that enhances cell adhesion or enhances the release of a substance from the cell. Examples of the coating material are poly (monostearoylglyceride co-succinic acid), poly-D, L-lactide-co-glycolide, sodium hyaluronate, n-isopropylacrylamide, collagen I to XIX, (Von Willebrand factor), osteopontin, fibrinogen, various elastin, various proteoglycans, various cardinalin, desmopressin, laminin-1 to 12, nitogen, tenacin, trombospondin, Choline, desmoglene, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, matrigel, poly-D-lysine, poly- Gel, various hydrogels, and cutting segments thereof. Here, two or more kinds of coating materials may be combined. It is also possible to use a medium used for culturing a carrier that supports cells and / or tissues on a surface and a polysaccharide such as guar gum, tamarind gum, locust bean gum, gum arabic, tara gum, tamarind gum, methyl cellulose, The above can also be mixed. The carrier may also contain a magnetic material, for example ferrite. The specific gravity of the carrier is preferably close to 1, more preferably 0.9 to 1.2, and particularly preferably about 1.0. Examples of carriers include, but are not limited to, Cytodex 1, Cytodex 3, Cytoline 1, Cytoline 2, Cytopore 1, Cytopore 2, (GE Healthcare Life Sciences), Biosilon TM (NUNC), Cultispher-G TM, Cultispher-S TM, Thermo SCIENTIFIC, HILLEXCT TM, ProNectinF-COATED TM And HILLEXII (registered trademark) (Solo Hill Engineering). The carrier may be sterilized as needed. The sterilization method is not particularly limited, and for example, radiation sterilization, ethylene oxide gas sterilization, autoclave sterilization and dry heat sterilization can be mentioned. A method for culturing animal cells using a carrier is not particularly limited, and a conventional culture method using a fluidized bed culture vessel or a packed bed culture vessel can be used. At this time, the carrier supporting the cells and / or the tissues on the surface and using the culture medium composition containing the nanofibers of the present invention enables uniform dispersion without even performing operations such as shaking. As a result, the desired cells and / or tissues can be cultured without loss of cell function. The cells and / or tissues cultured by this method can be collected by performing centrifugation and filtration treatment with the cells and / or tissue being supported by the carrier after incubation. In this case, after the used liquid medium is added, centrifugation and filtration treatment can be performed. For example, without limitation, the gravity acceleration (G) of the centrifugation is 100 G to 400 G, and the pore size of the filter used for filtration treatment is 10 탆 to 100 탆. Further, by encapsulating a material having magnetism such as ferrite in the carrier, the culture carrier can be recovered by the magnetic force. The cells and / or tissues cultured by this method can be collected by discharging from the carrier using various chelating agents, heat treatment or enzymes.

When cells and / or tissues are embedded in a carrier, a material composed of various polymers can be selected as a carrier. Examples of the polymer include collagen, gelatin, alginate, chitosan, agarose, polyglycolic acid, polylactic acid, fibrin adhesive, polylactic acid-polyglycolic acid copolymer, proteoglycan, glycosaminoglycan, polyurethane foam Poly-N-substituted acrylamide derivatives, poly N-substituted methacrylamide derivatives and copolymers thereof, polyvinyl methyl ether, polypropylene oxide, polyethylene oxide, partially acetylated < (Polyvinyl alcohol), methylcellulose, nitrocellulose, cellulose butyrate, polyethylene oxide, poly (2-hydroxyethyl methacrylate) / polycaprolactone, and the like, which are thermosensitive polymers such as poly Hydrogels may be mentioned. In addition, a carrier for embedding cells can be prepared by using two or more of these polymers. In addition, the carrier may have a physiologically active component in addition to such a polymer. Examples of physiologically active components include cell growth factors, differentiation inducing factors, cell adhesion factors, antibodies, enzymes, cytokines, hormones, lectins, extracellular matrices, and the like. In addition, one or more thickeners such as guar gum, tamarind gum, propylene glycol alginate ester, locust bean gum, gum arabic, tara gum and methyl cellulose may also be added to a medium used for culturing cells and / Can be mixed.

The method of embedding cells and / or tissues in these carriers is not particularly limited. For example, a method of aspirating a mixture of cells and the polymer into a syringe and dropping them from a needle of about 25 G to 19 G into a medium , Or dropping it onto a medium using a microwave pet, or the like can be used. The size of the bead-like carrier formed here is determined by the shape of the tip of the tool used when the mixture of the cell and the polymer is added dropwise, and it is preferably several tens of 탆 to several thousands of 탆, Is from 100 [micro] m to 2000 [micro] m. The number of cells that can be cultured on the beid-like carrier is not particularly limited, but can be freely selected depending on the bead size. For example, 5 million cells can be embedded in a bid-like carrier with a diameter of about 2000 占 퐉. Within the carrier, cells can be single dispersed or multiple cells can aggregate to form cell clumps. At this time, the carrier in which cells and / or tissues are embedded using the culture composition containing the nanofibers of the present invention can be evenly dispersed without performing operations such as stirring. As a result, the desired cells and / or tissues can be cultured without loss of cell function. The cells and / or tissues cultured by the present method can be collected by centrifugation and filtration treatment in which the cells and / or tissues are embedded in the carrier after incubation. In this case, after the used liquid medium is added, centrifugation and filtration treatment can be performed. For example, without limitation, the gravity acceleration (G) of the centrifugation is 100 G to 400 G, and the pore size of the filter used in the filtration treatment is from 10 μm to 100 μm. Cells and / or tissues cultured by this method can be collected by disintegrating the carrier by treatment with various chelating agents, heat, enzymes, etc., and dispersing them.

The method of forming cell aggregates (sphere) is not particularly limited and may be appropriately selected by those skilled in the art. Examples thereof include a method using a container having a cell non-attached surface, a hanging drop method, a swirl culture method, a three-dimensional scaffold method, a centrifugation method, a method using coagulation by an electric field or a magnetic field, and the like. For example, a target cell can be cultured in a culture container to which cell adhesion is inhibited by using a method using a container having a cell non-adhering surface to form a sphere. In the case of using the cell non-adherent culture container, first, the desired cells are collected, the cell suspension is prepared, and the culture is carried out by sowing in a culture container. If culturing continues for about one week, the cells spontaneously form a spear. As the cell non-adherent surface used herein, a surface of a culture container such as a commonly used chalet or the like (which is coated with a substance inhibiting cell adhesion, etc.) can be used. Examples of such materials are agarose, agar, poly-HEMA (poly- (2-hydroxy-ethyl methacrylate) 2-methacryloyloxyethylphosphoryl choline and other monomers such as butyl methacrylate Etc.). In the absence of cytotoxicity, the components are not limited to these.

As a method of forming cell aggregates (sphere), [NATURE BIOTECHNOLOGY, Vol. 28, NO. 4, APRIL 2010, 361-366, NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 689-700], [NATURE PROTOCOLS, VOL. 6, NO. 5, 2011, 572-579, Stem Cell Research, 7, 2011, 97-111], [Stem Cell Rev and Rep, 6, 2010, 248-259].

In addition, the medium used for culturing the sphere may also contain ingredients that promote the formation of sphere or promote its maintenance. Examples of the component having the above-mentioned effect include, but are not limited to, dimethyl sulfoxide, superoxide dismutase, carulloplasmin, catalase, peroxidase, L-ascorbic acid, L- ascorbic acid phosphate ester, tocopherol, flavonoid, , ROCK inhibitors such as selenium-containing compounds, transferrin, unsaturated fatty acids, albumin, theophylline, forskolin, glucagon, dibutyryl cAMP, Y27632, Fasudil (HA1077), H-1152 and Wf-536. Examples of selenium-containing compounds include sodium selenite, sodium selenate, dimethyl selenide, hydrogen selenide, selenomethionine, Se-methyl selenocysteine, selenocystionine, selenocysteine, selenohomocysteine, adenosine- -Triphosphoric acid, Se-adenosylselenomethionine can be mentioned. A plurality of depressions having the same diameter as the desired cell aggregate may also be introduced onto the cell non-adherent culture container to be used in order to obtain cell aggregates of a uniform size of interest. When these depressions are in contact with each other or within the diameter range of the desired cell aggregate and the cells are sown, the seeded cells do not form cell aggregates between the depressions, but certainly have a size corresponding to the volume in the depressions To obtain a population of cell aggregates of uniform size. The shape of the depressed portion at this time is preferably hemispherical or conical.

Alternatively, a support-based sphere exhibiting cell adhesion may also be formed. Examples of the support include collagen, polyrotaxane, polylactic acid (PLA), polylactic acid glycol (PLGA) copolymer, hydrogel and the like.

In addition, spores can also be formed by co-culturing with feeder cells. As feeder cells for promoting spear formation, any adherent cells can be used. Preferably, feeder cells for various cells are preferred. Examples of feeder cells include, but are not limited to, COS-1 cells and endothelial cells as preferred cell types, for example when spears of cells derived from liver or cartilage are formed.

In addition, a culture composition containing the nanofibers of the present invention may be used to form a sphere. In this case, it is necessary to add the nanofibers to the medium used for forming the sphere so that the concentration of the nanofibers becomes a concentration enabling suspension culture (preferably suspension culture) of the cells. For example, the concentration of the nanofibers is usually from 0.0001% to 1.0% (weight / volume), for example from 0.0005% to 1.0% (weight / volume), preferably from 0.001% to 0.3% , More preferably 0.005% to 0.1% (weight / volume), more preferably 0.01% to 0.05% (weight / volume) of the nanofibers in the medium used for forming spheres . The spear is prepared by uniformly dispersing the desired cells in a medium containing the nanofibers and allowing them to stand for 3 to 10 days to culture. The prepared sphere can be recovered by centrifugation and filtration treatment. For example, the gravity acceleration (G) of the centrifugal separation is 100 G to 400 G, and the size of the pores of the filter used for the filtration treatment is 10 탆 to 100 탆, but is not limited thereto. The cultured spores can be recovered by magnetic force using magnetic fine particles coated with an antibody that specifically binds to the target cells on the surface. Examples of such magnetic fine particles include Dynabeads (manufactured by Veritas Ltd.), MACS Micro Beads (manufactured by Miltenyi Biotec), BioMag (manufactured by Techno Chemicals Corporation), and the like.

The size of the sphere varies depending on the cell type and culture period, and is not particularly limited. When it is spherical or elliptical, its diameter is 20 탆 to 1000 탆, preferably 40 탆 to 500 탆, more preferably 50 탆 to 300 탆.

The spear can maintain the growth capacity for a period of 10 days or more, preferably 13 days or more, more preferably 30 days or more by continuing the stationary culture. By further performing mechanical splitting or single cell-forming treatment and aggregation periodically during the stationary culture, the proliferation capacity can be maintained substantially indefinitely.

The culture container used for culturing the sphere is not particularly limited as long as it allows culture of animal cells in general. For example, a flask, a dish, a chalet, a tissue culture dish, a multidish, a microplate, a microwell plate, a multiplate, a multiwell plate, a chamber slide, a chalet, a tube, a tray, a culture bag, .

The medium used for the culture of the spore may contain a cell attachment factor, and examples thereof include Matrigel, collagen gel, gelatin, poly-L-lysine, poly-D-lysine, laminin and fibronectin have. These cell adhesion factors may be added in combination of two or more. In addition, the medium used for culture of the spores can be mixed with thickeners such as guar gum, tamarind gum, propylene glycol alginate, locust bean gum, gum arabic, tara gum, methyl cellulose and the like.

By using the culture medium containing the nanofibers of the present invention, it is possible to provide a uniform dispersion even in a culture medium without performing an operation such as shaking. As a result, the desired cells and / or tissues can be cultured as spores without loss of cell function. The sphere cultured by this method can be collected by centrifugation or filtration treatment after culturing. In this case, after the used liquid medium is added, centrifugation or filtration treatment may be carried out. For example, the gravity acceleration (G) of centrifugal separation is 100 G to 400 G, and the size of the pores of the filter used in the filtration treatment is 10 탆 to 100 탆, but is not limited thereto. In addition, the magnetic microparticles coated on the surface by an antibody specifically binding to a target cell can be used to recover the sphere cultured by the magnetic force. Examples of the magnetic fine particles include Dynabeads (manufactured by Veritas Ltd.), MACS Microbiide (manufactured by Miltenyi Biotec), BioMag (manufactured by Techno Chemicals Corporation) and the like. The recovered sphere may be dispersed as a single cell by further decomposition by treatment with various chelating agents, heat, filters, enzymes and the like.

As a method for culturing the cells and / or tissues derived from plants, a unified tissue that is an aggregate of undifferentiated plant cells can be cultured. Fusion tissue can be derived by known methods for each plant species used. For example, the surface of some of the tissues of differentiated plants (for example, roots, stems, leaf parts, seeds, growth spots, pears, pollen, etc.) may be treated with 70% alcohol, 1% sodium hypochlorite solution (For example, about 1 to about 5 mm square area) using a knife or the like, sterilizing the tissue portion by aseptic operation using a clean laboratory bench, etc., Inoculate the induction medium and cultivate under appropriate conditions. The derived union tissue can be applied to the liquid culture for mass proliferation or can be maintained as a seed strain by subculturing in a passage medium. The subculture may be carried out using any of liquid medium and solid medium.

The amount of the plant cell aggregate to be inoculated at the time of initiating the stationary culture using the medium composition of the present invention can be appropriately determined depending on the growth rate of the target cell, the culture method (batch culture, feed-batch culture, And the like. For example, when a plant cell aggregate such as a fusion tissue is to be cultured, the wet weight of the cell aggregate relative to the culture medium composition of the present invention is 4 to 8% (w / v), preferably 5 to 7% (w / v)% of the culture medium. The particle size of the plant cell aggregate during incubation is from 3 mm to 40 mm, preferably from 3 mm to 20 mm, more preferably from 5 mm to 15 mm. As used herein, the term "particle diameter" means, for example, the diameter when the plant cell aggregate is spherical, and the long axis if it is an elliptical spherical shape,

The temperature at which the cells and / or tissues are cultured is generally 25 to 39 DEG C, preferably 33 to 39 DEG C for animal cells. The CO ₂ concentration is usually 4 to 10% by volume and preferably 4 to 6% by volume in the culture atmosphere. The incubation period is usually from 3 to 35 days, and can be set freely according to the purpose of culturing. The cultivation temperature of plant cells is usually 20 to 30 DEG C, and when light is required, it can be cultured under illumination conditions of 2000 to 8000 lux. The incubation period is usually 3 to 70 days, but it can be set freely according to the purpose of the incubation.

When cells and / or tissues are cultured by the method of the present invention, separately prepared cells and / or tissues are added to the culture composition of the present invention and mixed to produce a uniform dispersion. In this case, the mixing method is not particularly limited, and examples thereof include mixing using devices such as manual mixing using a pipetting or the like, a stirrer, a vortex mixer, a microplate mixer, and a shaking machine. After mixing, the culture medium can be kept stationary, or the culture medium can be rotated, shaken, or agitated, as needed. The number of revolutions and the frequency can be appropriately set according to the purpose of the person skilled in the art. When the culture medium composition needs to be exchanged during the stationary culture period, the cells and / or tissues and the culture medium composition can be separated by centrifugation or filtration treatment, and a new culture composition can be added to the cells and / or tissues. Alternatively, cells and / or tissues can be suitably concentrated by centrifugation or filtration treatment, and a new medium composition can be added to the concentrate. For example, without limitation, the gravity acceleration (G) of the centrifugation is 100 G to 400 G, and the size of the pores of the filter used for the filtration treatment is 10 탆 to 100 탆. In addition, the cultured cells and / or tissues can be separated by a magnetic force using magnetic fine particles coated on the surface by an antibody specifically binding to a target cell. Examples of the magnetic fine particles include Dynabeads (manufactured by Veritas Ltd.), MACS Microbiide (manufactured by Miltenyi Biotec), BioMag (manufactured by Techno Chemicals Corporation) and the like. The exchange of the medium composition can also be carried out by means of a bioreactor and an automatic culture apparatus which can be carried out under mechanical control and under a closed environment.

[Method of preserving or transporting cells or tissues]

In addition, the present invention provides a preservation method and a transportation method for preserving cells or tissues using the above-mentioned medium composition of the present invention. In the preservation or transportation method of the present invention, the cells and tissues can be stored (preferably in a suspended state), and preserved or transported, by using the culture medium composition of the present invention.

Cells and tissues targeted for storage or transportation include those described above as cells and tissues that can be used for culture using the medium composition of the present invention.

In addition to the above-mentioned composition, the culture medium composition of the present invention used for preservation or transportation may contain various components having cell-life-lengthening effect upon preservation of cells and tissues in a non-frozen state. Examples of the components include saccharides (except for polysaccharides) (e.g., monosaccharides, disaccharides), antioxidants (e.g., SOD, vitamin E or glutathione), hydrophilic polymers (e.g., polyvinylpyrrolidone Chelating agents (e.g., EDTA), sugar alcohols (e.g., mannitol, sorbitol), glycerol, and the like.

In the preservation or transportation method of the present invention, the desired cells or tissues are dispersed in the medium composition of the present invention and placed in a sealable container. Examples of the container include, but are not limited to, a flask, a plastic pack, a Teflon (registered trademark) bag, a tube, and a culture bag. In order to avoid contamination of the contents or the contamination of the bacteria or the like from the outside during storage or transportation, the container into which the dispersion of cells and tissues is put in the culture medium composition of the present invention is preferably sealed.

The temperature during preservation or transportation is not particularly limited as long as the survival of cells or tissues is maintained, but is usually 37 DEG C or lower. When the temperature is low, deterioration of cell or tissue viability during storage or transportation can be avoided. However, in order to prevent freezing of cells or tissues, they are usually stored or transported at a temperature exceeding the melting point of the medium composition of the present invention. Therefore, the temperature during storage or transportation is usually maintained at -5 to 42 캜, preferably 1 to 37 캜, more preferably 4 to 32 캜, and still more preferably 18 to 30 캜.

In order to enable preservation or transportation of the cells or tissues in the suspended state, the temperature during the preservation or transportation is preferably a temperature that enables the suspension of the cells or tissues in the culture medium composition of the present invention. The temperature at which the cells or tissues can be suspended can be appropriately set according to the kind of the raw material constituting the nanofibers.

In one embodiment, carrageenan (preferably, kappa-carrageenan) is used as a raw material for composing the nanofiber contained in the medium composition of the present invention used in the preservation or transportation method of the present invention. The culture medium composition of the present invention containing nanofibers composed of carrageenan has a suspension action at 25 占 폚 or lower and loses its suspension action at 37 占 폚. Accordingly, the desired cells or tissues are stored or transported at a temperature of 25 占 폚 or lower (preferably 0-25 占 폚), and after the completion of storage or transportation, the temperature is maintained at 37 占 폚 or higher Deg.] C, preferably 37 [deg.] C), cells or tissues can be easily recovered by precipitating suspended cells or tissues.

The period of preservation or transportation is not particularly limited within the range in which the cells or tissues can be maintained in a viable state in the medium composition of the present invention. This is usually 1 hour or more and 10 days or less, preferably 1 to 8 days, more preferably 1 to 3 days. During storage or transportation, it is preferred that the cells or tissues remain in a suspended state in the medium composition of the present invention.

Using the preservation or transportation methods of the present invention, cells and tissues can be maintained in suspension. Therefore, it is possible to avoid damage to cells and tissues caused by separation of cells from the plate due to vibration during transport or aggregation of tissues and tissues in contact with each other due to sedimentation, And tissue can be preserved and transported.

[Example]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to examples of the medium composition of the present invention. The materials, the amounts to be used, the ratios, the processing contents, and the processing procedures shown in the following examples can be suitably changed as long as they do not depart from the gist of the present invention. Therefore, the scope of the present invention should not be construed as being an embodiment shown below.

Reference Example 1 Viscosity measurement and cell suspension test of a medium containing deacylated gellan gum heat-treated at a high temperature

Preparation and viscosity measurement of deacylated gellan gum-containing medium

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in pure water at 0.4% (w / v) and dissolved with stirring while heating at 90 ° C. This aqueous solution was cooled to room temperature with stirring and sterilized in an autoclave at 121 占 폚 for 20 minutes. (DMEM / F-12 medium, 50 mL) and sterilized water (47.5 mL) were placed in a 300-mL tall beaker while stirring at room temperature with a homomixer (3000 rpm) An aqueous gellan gum solution (2.5 mL) was added and the mixture was kept stirred for 1 minute to prepare a deacylated gellan gum medium composition to a final concentration of 0.01%. A media composition was similarly prepared to which an aqueous deacylated gellan gum solution with final concentrations of 0.02, 0.03, and 0.05% (w / v) was added. The viscosity of the culture medium composition was measured using an E-type viscometer (Viscometer TVE-22 L, standard rotor 1 ° 34 '× R24, manufactured by Toki Sangyo Co., Ltd.) at 100 rpm under the condition of 37 ° C. for 5 minutes .

Cell suspension of deacylated gellan gum-containing medium

The human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) Was suspended in EMEM medium (WAKO) containing 10% (v / v) fetal calf serum at 250,000 cells / mL, Was inoculated on EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) And cultured in a CO ₂ incubator (5% CO ₂ ) for 3 days. The suspension (10 mL) of the obtained spheres (100 to 200 mu m in diameter) was centrifuged (200 G, 5 minutes) to precipitate the spores and remove the supernatant to prepare a sphere suspension (1.0 mL). Subsequently, the prepared deacylated gellan gum-containing medium was added to 1.0 mL of a 1.5 mL eppendorf tube, and an additional HeLa cell sphere suspension (10 μL) was added. The cell aggregates were dispersed by tapping, incubated at 37 DEG C, and after one hour, the dispersion state of the cells was visually observed.

[Table 1]

Reference Comparative Example Preparation of methylcellulose and collagen-containing medium

Preparation of methylcellulose-containing medium

To the 200 mL recovery flask was added DMEM / F-12 medium (100 mL, manufactured by Aldrich), and methylcellulose (M0387, 0.1 g, manufactured by Aldrich) was added. The mixture was stirred while cooling in an ice bath to dissolve methylcellulose. Using this solution, a medium composition was prepared in which a methyl cellulose aqueous solution was added at a final concentration of 0.1, 0.3, 0.6 or 1.0% (w / v).

Preparation of collagen-containing medium

(DMEM / F-12 medium (1 mL, manufactured by Aldrich), reconstitution buffer (1 mL, manufactured by Nitta Gelatin Inc.), and 10 mL of a 10-fold concentration of a cell matrix type IA (manufactured by Nitta Gelatin Inc., Water (1.5 mL) was added and the mixture was stirred in ice to make a 0.2% collagen-containing medium. Similarly, a media composition was prepared in which collagen was added at a final concentration of 0.01, 0.05, 0.1, or 0.2% (w / v).

The prepared medium composition was applied to the suspension test and viscosity measurement of HeLa cell sphere in the same manner as the deacylated gellan gum-containing medium. The viscosity of 1.0% (w / v) methylcellulose was measured at 50 rpm due to the measuring range of the apparatus.

[Table 2]

[Table 3]

Reference Experiment Example

In the following Reference Experimental Example, the concentration (%) of CO _{2 in} the CO ₂ incubator is represented by the volume percentage of CO _{2 in} the atmosphere. PBS means phosphate buffered physiological saline (Sigma Aldrich Japan), and FBS means fetal calf serum (Biological Industries). (W / v) represents the weight per volume.

Reference Example 1: Cell proliferation test by dispersing single cells

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) at 0.3% (w / v) and dissolved with stirring while heating at 90 캜. This aqueous solution was sterilized in an autoclave at 121 캜 for 20 minutes. This solution was used to remove deionized water in a final concentration of 0.015% (w / v) in an IMDM medium (manufactured by Gibco) containing 10% (v / v) fetal calf serum and 10 ng / mL of thrombopoietin And a true culture gellan gum was added thereto to prepare a culture medium composition. Subsequently, the human leukemia cell line UT7 / TPO was inoculated into a medium composition to which 20,000 cells / mL of the above-mentioned deacylated gellan gum had been added, and 5 mL / well of 6-well flat microplate (manufactured by Corning Incorporated) ≪ / RTI > Similarly, a human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) Was inoculated into EMEM medium (WAKO) containing 10% (v / v) fetal calf serum with 0.015% (w / v) The seeds were seeded at 20,000 cells / mL in a culture medium obtained by adding gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.), and seeded in 6-well flat microplates (manufactured by Corning Incorporated) ≪ / RTI > The cell suspension was incubated for 3 days in a CO ₂ incubator (5% CO ₂ ) while standing still. Then, a part of the culture medium was recovered, and the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added, and the number of viable cells was measured by a blood cell meter (ERMA INC.).

As a result, it was confirmed that UT7 / TPO cells and HeLa cells can be uniformly cultured in a suspended state using the above-mentioned medium composition, and efficiently proliferated in the medium composition. The cell numbers of UT7 / TPO cells and HeLa cells after 3 days of political suspension cultivation are shown in Table 4.

[Table 4]

Reference Example 2: Cell proliferation test by culturing cell line-derived spear

The human liver cancer cell line HepG2 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) Was suspended in DMEM medium (WAKO) containing 10% (v / v) fetal calf serum at 250,000 cells / mL, Was inoculated on EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) And cultured in a CO ₂ incubator (5% CO ₂ ) for 7 days. Similarly, human cervical cancer cell line HeLa (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) Was suspended in EMEM medium (WAKO) containing 10% (v / v) fetal calf serum at 250,000 cells / mL, (10 mL) was inoculated on EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) And cultured in a CO ₂ incubator (5% CO ₂ ) for 7 days. The suspension (2.5 mL) of each of the obtained cell lines (100 to 200 mu m in diameter) was centrifuged (200 G, 5 minutes) to precipitate the spores, and the supernatant was removed. Subsequently, the above-mentioned medium (10 mL) was added to a spear (about 800 spears), and the suspension was transferred to a pancake tube (manufactured by BM Equipment Co., Ltd.). Similarly, a sphere suspension was prepared using the medium composition obtained by adding 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) to the above-mentioned medium , And then transferred to a flat tube (manufactured by BM Equipment Co., Ltd.). The culture medium to which 0.015% (w / v) of the deacylated gellan gum was added was prepared by first adding deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) to 0.3% (w / v) -Q water), which was stirred and dissolved while heating at 90 DEG C, and this aqueous solution was sterilized in an autoclave for 20 minutes at 121 DEG C and contained 10% (v / v) fetal calf serum by 1/20 dilution Lt; RTI ID = 0.0 > DMEM < / RTI >

The above-mentioned spear suspension was allowed to stand still in a CO ₂ incubator (5% CO ₂ ) at 37 ° C for 3 days, and then a medium of double volume was added. The mixture was centrifuged (200 G, 5 min) to settle the spear and the supernatant was removed. At this point, a part of the sphere was taken and its shape was observed with an optical microscope (OLMPUS, CK30-F100). Subsequently, the recovered sphere was washed once with PBS (10 mL), 1 mL of tryptic-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C for 5 minutes. The above-mentioned medium (9 mL) was added and cells were collected by centrifugation (200 G, 5 min). An aliquot of the obtained cell suspension (2 mL) was added in the same amount as a tryptic blue staining solution (manufactured by Invitrogen Corporation), and the number of viable cells and dead cells was measured by a hemocytometer (ERMA INC. .

As a result, it was confirmed that the spear of HepG2 cells and HeLa cells could be cultured in suspension using the above-mentioned medium composition, and the cells proliferated efficiently in the medium composition. In addition, it was confirmed that when the culture medium was grown, the ratio of the cells was smaller than that of the conventional culture medium, and that the culture medium had an excellent cell proliferation promoting effect. The spear cultured in the existing medium is settled in the lower part of the culture container. Further, the shape of the cultured sphere was observed with an optical microscope. As a result, the culture medium did not show association of spears, whereas association of spears was observed in existing media.

Table 5 shows the relative numbers of HepG2 cells and HeLa cells with the number of cells cultured in the medium containing no deacylated gellan gum. Table 6 shows the relative cell death rate of cells cultured in a medium containing no deacylated gellan gum (number of cells / number of cells). Suspensions of spermatozoa of HepG2 cells and HeLa cells cultured in the medium composition are shown in Figs. 1 and 2, respectively. The shape of the sphere of the cultured HeLa cells is shown in Fig.

[Table 5]

[Table 6]

Reference Example 3: Cell proliferation test by culturing cell line adhered on micro carrier

Microcarrier Cytodex (R) 1 (GE Healthcare Life Sciences) was suspended in PBS at 0.02 g / mL and the suspension was allowed to stand overnight. The supernatant was discarded and the microcarrier was washed twice with fresh PBS. Thereafter, it was suspended again in PBS at 0.02 g / mL and sterilized in an autoclave at 121 DEG C for 20 minutes. Subsequently, these microcarriers were washed twice with 70% ethanol and three times with PBS, and suspended in DMEM medium (WAKO) containing 10% (v / v) fetal calf serum at 0.02 g / mL. Using this microcarrier suspension, 120 mg of Cytodex (R) 1 and 4,000,000 HepG2 cells in DMEM medium (containing 10% (v / v) fetal calf serum, 20 mL) were prepared and the cell suspension (100 rpm) in a beaker treated with a silicone coating agent (manufactured by AGC TECHNO GLASS Co., Ltd.) for 6 hours at 37 캜 using a stirrer. At this point, adhesion of HepG2 cells to the microcarriers was confirmed by microscopy. Subsequently, the cell-attached microcarriers were washed twice with DMEM medium containing 10% (v / v) fetal calf serum and suspended in the same medium (3 mL).

DMEM medium (20 mL) containing 10% (v / v) fetal calf serum and 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) (300 μL) was added to each of the above-obtained medium compositions and cultured at 37 ° C for 3 days. For cultures containing no deacylated gellan gum, the mixture was incubated with agitation (100 rpm) on a stirrer. After the incubation, the attachment state of the cells on the micro carrier was confirmed by a microscope, and the micro carrier was precipitated by centrifugation (200 G, 5 minutes). This microcarrier was washed with PBS (10 mL), 1 mL of tryptic-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C for 5 minutes. In addition, DMEM medium (9 mL) containing 10% (v / v) fetal calf serum was added and the microcarriers were removed using a Cell Strainer (manufactured by BD Falcon, mesh size 70 μm). Cells were recovered from the obtained filtrate by centrifugation (200 G, 5 minutes). The cells were suspended in a medium (500 μL), and a part thereof was added with the same amount of tryptic blue staining solution (manufactured by Invitrogen Corporation), and the number of viable cells was measured with a hemocytometer (manufactured by ERMA INC.). As a result, the culture without deacylated gellan gum contained 123,000 cells, while the culture medium containing the deacylated gellan gum contained 1,320,000 cells. As mentioned above, it has been confirmed that the culture medium composition containing the specific compound structure is superior to the conventional culture medium in promoting cell proliferation even when cells are cultured using a micro carrier. The adhesion state of HepG2 cells after 3 days of microcarrier culture using the culture composition containing the specific compound structure is shown in Fig.

Reference Example 4: Cell suspension test using cell line-derived spear

Zincan gum (KELTROL CG, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) at a concentration of 1% (w / v) and dissolved with stirring while heating at 90 캜. Using this aqueous solution, a DMEM / F-12 medium composition having a final concentration of xanthan gum of 0.1, 0.15, or 0.2% (w / v) was prepared. In addition, a mixture of 0.2% (w / v) k-carrageenan (GENUGEL WR-80-J manufactured by SANSHO Co., Ltd.) and 0.2% (w / v) locust bean gum GENUGUM RL- Ltd.) was heated at 90 占 폚 to prepare an aqueous solution. Using this aqueous solution, a composition of DMEM / F-12 medium (manufactured by Sigma Ltd.) containing 0.03, 0.04 or 0.05% (w / v) of k-carrageenan and locust bean gum was prepared.

In the same manner as in Experimental Example 2, a sphere of HeLa cells was formed, dozens of sphere were added to each of the prepared medium (1 mL), the mixture was allowed to stand at 37 캜 for 1 hour, The suspended state was visually observed. As a result, the sphere of the HeLa cells was maintained in a suspension state in any of the above-mentioned medium composition. Addition of an equal volume of medium to the cell suspension and its centrifugation (300-400 G, 5 minutes) also confirmed precipitation and recovery of sphere of HeLa cells. The suspended state of the sphere of HeLa cells cultured in the medium composition is shown in Fig. 5, respectively. In addition, the viscosities measured in the same manner as in Analysis Example 1 are shown in Tables 7 and 8.

[Table 7]

[Table 8]

Reference Example 5: Cell suspension test using filter medium-filtered medium composition

In the same manner as in Reference Example 2, DMEM / F-12 medium composition containing 0.015% of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was prepared. Subsequently, this medium composition (1 mL) was dispersed in a 70 탆 filter or 40 탆 filter (BD Falcon), 30 탆 filter or 20 탆 filter (AS ONE Corporation), 10 탆 filter (Partec) Filter, 1.2 占 퐉 filter, 0.45 占 퐉 filter or 0.2 占 퐉 filter (Sartorius Stedim Japan). Spheres of HepG2 cells prepared in the same manner as in Reference Example 2 were added to about 10 sphere for the above-mentioned filtrate, allowed to stand at 37 캜 for 1 hour, and the suspension state of the sphere cells was visually observed did. As a result, it was confirmed that the sphere of HepG2 cells was maintained in suspension in a medium composition having passed through a filter having a size of 10 mu m or more, but was settled in a medium composition passed through a filter of 5 mu m or less. Further, centrifugation at a room temperature, 300 G, 5 minutes, or an equivalent volume of the medium of the HepG2 cell sphere in a suspension state and centrifugation at room temperature, 200 G, 5 minutes caused sedimentation and recovery of the spore Confirmed.

Reference Example 6: Spear forming test

EMEM medium (WAKO) containing 0.01% of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v / v) fetal serum was obtained in the same manner as in Reference Example 2, ≪ / RTI > Subsequently, HeLa cells were added at a concentration of 1000 cells / mL and distributed in 24-well plates (manufactured by Corning Incorporated). These plates were allowed to stand-culture at 37 DEG C for 9 days, and the formation of sphere was confirmed by a microscope. The spear cells were sedimented by centrifugation treatment (300 G, 5 minutes) and washed once with PBS (5 mL). 100 μL of tryptic-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C. for 5 minutes. Here, EMEM medium (100 μL) containing 10% (v / v) fetal serum was added to the resulting cell suspension (100 μL), and a trypsin blue staining solution (Invitrogen Corporation) , And the number of viable cells was measured by a hemocyte calculator (manufactured by ERMA INC.). As a result, it was confirmed that HeLa cells were increased by 170,000 cells / mL. The sphere of HeLa cells formed in the medium composition is shown in Fig.

Experimental Example 7: Optical Microscope Observation of Structure

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in pure water at 0.4% (w / v) and dissolved with stirring while heating at 90 ° C. DMEM / F-12 medium (95 mL, manufactured by Aldrich) was added to a 300-mL toll beaker, and an aqueous deacylated gellan gum solution (5 mL) was added thereto while stirring with a magnetic stirrer at room temperature. Was continued to prepare a culture composition containing deacylated gellan gum at a final concentration of 0.02%. Further, the medium composition was stirred for 5 minutes by a homomixer (3000 rpm). The prepared culture medium composition was observed with an optical microscope (KEYENCE Corporation, BIOREVO BZ-9000). The observed structure is shown in Fig.

Experimental Example 8: Production by mixed heating of powder medium and DAG

(20 mg) and DMEM / F-12 medium (1.58 g, manufactured by Life Technologies) were placed in a 200 mL Erlenmeyer flask, and 100 mL of pure water (100 mL) . The mixture was sterilized in an autoclave at 121 캜 for 20 minutes to prepare a DMEM / F-12 medium composition having a deacylated gellan gum concentration of 0.02%. Dextranide Cytodex 1 (200 쨉 m in size, manufactured by GE Healthcare Life Sciences) was added to the prepared medium to visually confirm the bead dispersion state. For evaluation, the dispersed state is?, The sedimented / dispersed state is?, And the sedimentary state is x. The results are shown in Table 9 below.

[Table 9]

Reference Example 9: Preparation of a medium composition containing a polysaccharide

Xanthan gum (KELTROL CG, manufactured by SANSHO Co., Ltd.) was suspended in pure water at a concentration of 0.5% (w / v) and dissolved by stirring while heating at 90 캜. Similarly, sodium alginate (Duck alginic acid NSPM, manufactured by FOOD CHEMIFA Co., Ltd.), locust bean gum (GENUGUM RL-200-J, manufactured by SANSHO Co., Ltd.), k- Aqueous solution of 0.5% (w / v) of diethanolamine (manufactured by SANSHO Co., Ltd.) or dianthum gum (KELCO CRETE DG-F, manufactured by SANSHO Co., Ltd.)

Aqueous solution and a 0.2 or 0.1% (w / v) deacylated gellan gum solution and a 10-fold concentration of DMEM / F-12 medium were each mixed and the mixture was heated at 80 ° C for 30 minutes, cooled to room temperature, A solution of sodium bicarbonate in DMEM / F-2 was added to the final concentration of 0.01, 0.02% (w / v) of deacylated gellan gum and other polysaccharides at final concentrations of 0.1, 0.2, 0.3, 0.4% (w / v) 12 medium composition was prepared. In addition, a medium containing the deacylated gellan gum was prepared as mentioned above, and a powder of methyl cellulose (cP400, manufactured by WAKO) was added. The mixture was stirred in an ice bath to dissolve the methylcellulose and the other methylcellulose was added to the final concentration of 0.01, 0.02% (w / v) with deacylated gellan gum and final concentrations of 0.1, 0.2, 0.3, 0.4% (w / v) Containing DMEM / F-12 medium was prepared.

Polystyrene beads (size 500 - 600 μm, manufactured by Polysciences Inc.) were added to the prepared culture medium to visually confirm the dispersion state of the beads. For the evaluation, the suspended state is?, The sedimentary / dispersed state is?, And the sedimentary state is x. The results are shown in Table 10.

[Table 10]

Reference Example 10: Viscosity measurement of a medium composition containing polysaccharides

DMEM / F-12 medium containing deacylated gellan gum and other polysaccharides was prepared at a final concentration of 0.005, 0.01% (w / v) by a method similar to that for the polysaccharide mixture of Reference Example 9. The final concentrations of polysaccharides were 0.1% w / v for xanthan gum, sodium alginate, locust bean gum, 0.2% w / v for methylcellulose, 0.05% w / v). The viscosities of each of the media compositions and measured by a method similar to that in Analysis Example 1 are shown in Tables 11 to 16.

[Table 11]

[Table 12]

[Table 13]

[Table 14]

[Table 15]

[Table 16]

Experimental Example 11: Preparation of Medium Composition Having Modified Divalent Metal Ion Concentration

Using 0.02% (w / v) of deacylated gellan gum in the same manner as in Reference Example 8 using DMEM / F-12 (D9785, manufactured by Aldrich) containing no calcium chloride, magnesium sulfate and magnesium chloride, Gt; DMEM / F-12 < / RTI > A DMEM / F-12 medium composition was prepared by adding calcium chloride or magnesium sulfate and magnesium chloride so that the final concentration was set to a defined amount of DMEM / F-12 medium. From the viewpoint of the defined composition of the DMEM / F-12 medium, the final concentration of each was set at 0.116 g / L for calcium chloride, 0.049 g / L for magnesium sulfate and 0.061 g / L for magnesium chloride.

Dextranide Cytodex 1 (manufactured by GE Healthcare Life Sciences) was added to the prepared culture medium composition, and after 2 days, the state of dispersion of the beads was confirmed by visual observation. For the evaluation, the suspended state is?, The sedimentation / dispersion state is?, And the sedimentation state is x. The results are shown in Table 17.

[Table 17]

Reference Example 12: Preparation of a medium composition to which a divalent metal cation was added later

(DMEM / F-12 medium (containing no calcium chloride, magnesium sulfate and magnesium chloride, D9785, manufactured by Aldrich), calcium chloride (1167 mg) in a concentration of 0.1% (w / v) , Magnesium sulfate (489 mg) and magnesium chloride (287 mg) were dissolved in pure water (300 mL) to prepare a salt solution. An aqueous solution of deacylated gellan gum and a pure water were added to a 200 mL toll beaker, and the solution was stirred at 200 rpm using an anchor type stirring blade. Solution A, which is a mixture of the medium solution and water, was added and the mixture was immediately stirred for 10 minutes. Thereafter, the salt solution was added, and a 7.5% aqueous solution of sodium bicarbonate (1.6 mL) was further added to prepare a DMEM / F-12 medium composition containing the deacylated gellan gum at a final concentration of 0.02%. The mixing amount of each solution is shown in the table. After 4 hours of manufacture, 6 media compositions were applied to the dispersion evaluation of polystyrene beads and Cytodex1. The results are shown in Tables 18 and 19.

[Table 18]

[Table 19]

Reference Example 13: Preparation of various medium composition

0.1% (w / v) deacylated gellan gum solution and a high concentration of the medium solution were prepared. RPMI-1640 (R6504, manufactured by Aldrich) and DMEM (corresponding to high-pressure sterilization, manufactured by Nissui) were prepared at a concentration of 10 times as high as the concentration of MEM (M0268, manufactured by Aldrich). 0.1% (w / v) deacylated gellan gum solution, each of the high concentration medium and pure water for concentration adjustment, was mixed and heated at 80 DEG C for 30 minutes. The mixture was cooled to room temperature and 7.5% aqueous sodium bicarbonate solution was added to prepare a culture composition containing the deacylated gellan gum at final concentrations of 0.01, 0.02, 0.03% (w / v). The six media compositions thus prepared were evaluated for the suspension and dispersion state of the polystyrene beads and dextran beads Cytodex 1, where the suspension state is?, Some sedimentation / dispersion states are?, And the sedimentation state is 占. The results are shown in Tables 20 and 21.

[Table 20]

[Table 21]

Reference Example 14: Measurement of particle size distribution of a culture composition containing deacylated gellan gum

According to Reference Example 1, a DMEM / F-12 medium composition containing 0.038% (w / v) of deacylated gellan gum was prepared. The medium was prepared by stirring with a homomixer at 3000 rpm or 6000 rpm for 1 minute. The particle size distribution of the media composition was measured using a Beckman Instruments Coulter, Inc. And the median diameter (d50) of the volume standard particle size distribution was measured by Multisizer 4 (a device for accurately measuring the particle size distribution by the Coulter's theorem). The results are shown in Table 22.

[Table 22]

Reference Example 15: Phosphorylation of deacylated gellan gum

In a 100 mL glass test tube, deacylated gellan gum (1 g) and pure water (40 mL) were weighed and the mixture was heated at 100 占 폚 for 30 minutes to prepare a suspension. To this suspension was added an aqueous phosphoric acid solution (85%, 1 g) and the mixture was heated under reflux for 5 hours. It was then cooled to room temperature with stirring for 12 hours and the resulting white suspension was taken up in 99% ethanol (500 mL). The resulting floc white solid was collected by filtration and dried to give a light brown solid (0.4 g) as a phosphorus oxide of deacylated gellan gum. The introduction of the phosphate group was confirmed by Fourier-transform infrared spectroscopic analysis (1700 cm-1; P-OH, 1296 cm-1, 1265 cm-1; P = O) by IR-Prestage 21 manufactured by SHIMADZU CORPORATION. The light brown solid was decomposed by a microwave heating decomposition apparatus (ETHOS TC, manufactured by Milestone General) and the content of phosphorus atoms was measured by ICP-OES (SPS 5520, manufactured by SII NanoTechnology). The result was 3.5 wt% (n = 2).

Reference Example 16: Preparation of a culture medium composition containing phosphorylated deacylated gellan gum

An amount of phosphorylated deacylated gellan gum (30 mg) and DMEM / F-12 medium (1.56 g, manufactured by Life Technologies) were placed in a 200 mL Erlenmeyer flask, and pure water (100 mL) was poured therein. The mixture was sterilized in an autoclave at 121 占 폚 for 20 minutes to prepare a DMEM / F-12 medium composition having a deacylated gellan gum concentration of 0.03%. Dextranide Cytodex 1 (manufactured by GE Healthcare Life Sciences) was added to the prepared medium, and the dispersed state of the beads was confirmed by visual observation. Phosphorylated deacylated 0.03% (w / v) gellan gum concentration revealed the bead dispersion.

Reference Example 17: Preparation of a culture medium composition containing deacylated gellan gum

DMEM / F-12 medium composition having a deacylated gellan gum concentration of 0.02% was prepared by mixing the deacylated gellan gum aqueous solution and the medium solution at the ratios shown in the following table, and polystyrene beads (Size 500-600 μm, Polysciences Inc Ltd.) was evaluated. The results are shown in Tables 23 and 24. Styreneide was dispersed under all conditions by standing for more than one day.

[Table 23]

"DMEM / F12 powder medium / pure water" was added to "deacylated gellan gum / pure water ".

[Table 24]

"Deacylated gellan gum / pure water" was added to "DMEM / F12 powder medium / pure water ".

Reference Example 18: Preparation of medium composition using filter

Dehydrated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) at a final concentration of 0.02 or 0.04% (w / v) Lt; 0 > C for 20 minutes. Further, this aqueous solution (100 mL) was filtered through a polyether sulfone membrane filter (manufactured by Corning Incorporated) having a pore size of 0.22 탆. Subsequently, the filtrate was mixed with DMEM / F-12 medium (Sigma Aldrich) at 2- to 4- fold concentration, and the mixture was treated with a mild mixer (SI-24, TAITEC Co., Ltd.) (For example, 0.02% (w / v) of deacylated gellan gum aqueous solution and 2-fold concentration of dextrinized gellan gum at a final concentration of 0.01 or 0.015% (w / v) DMEM / F-12 medium were mixed at 25 mL each to prepare a 0.01% (w / v) deacylated gellan gum medium composition (50 mL). A spear of HepG2 cells was formed by a method similar to that in Reference Example 2, and several tens of spears were added to the prepared medium (1 mL), and the mixture was allowed to stand at 37 DEG C, and after 1 hour and overnight, The suspended state was visually observed. As a result, it was confirmed that the sphere of HepG2 cells was maintained in a suspension state in all of the above-mentioned medium compositions. In addition, twice the volume of the medium was added, and the cell suspension was centrifuged (500 G, 5 minutes). It was confirmed that the sphere of HepG2 cells precipitated and the cells could be recovered from all the medium compositions. After one night, the dispersion state of the spear was confirmed and evaluated by visual observation, wherein the suspended dispersion state was O, the sedimentation / dispersion state was Δ, and the sedimentation state was ×. The evaluation results are shown in Table 25.

[Table 25]

Reference Example 19: Cell proliferation test by culturing cell line-derived spear

The human fetal kidney cell line HEK293 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) Was suspended in EMEM medium (WAKO) containing 10% (v / v) fetal calf serum at 250,000 cells / mL, ) Was inoculated on EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) And cultured in a CO ₂ incubator (5% CO ₂ ) for 2 days. The suspension (10 mL) of the spores (100 to 200 mu m in diameter) of HEK293 cells thus obtained was centrifuged (200 G, 5 minutes) to precipitate the spores, remove the supernatant, and suspend the spear in 1 mL. Subsequently, the medium (10 mL) was added to a sphere suspension (200 μL, cell number: about 200,000), and the suspension was transferred to a pouch tube (manufactured by BM Equipment Co., Ltd.). Similarly, a sphere suspension was prepared using the medium composition obtained by adding 0.015% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) to the above-mentioned medium , And transferred to a flat tube (manufactured by BM Equipment Co., Ltd.). The culture medium to which 0.015% (w / v) of deacylated gellan gum had been added was first diluted with 0.3% (w / v) of deacylated gellan gum (KELCOGEL CG-LA, SANSHO Co., Water) and dissolving by stirring with heating at 90 DEG C, the aqueous solution is sterilized in an autoclave at 121 DEG C for 20 minutes, and the solution is diluted 1/20 in 10% (v / v) To EMEM medium containing serum.

After the stationary culture of the above-mentioned spear suspension in a CO ₂ incubator (5% CO ₂ ) for 5 days at 37 ° C, twice the volume of medium was added. The mixture was centrifuged (500 G, 5 min) to precipitate the spores and the supernatant was removed. Subsequently, the recovered sphere was washed once with PBS (10 mL), 1 mL of tryptic-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C for 5 minutes . The above-mentioned medium (9 mL) was added and cells were collected by centrifugation (500 G, 5 min). A portion of the resulting cell suspension (2 mL) was added in the same amount as a trypan blue staining solution (manufactured by Invitrogen Corporation), and the number of living cells and the number of dead cells was measured with a hemocyte calculator (manufactured by ERMA INC.). As a control, a culture composition without deacylated gellan gum was prepared and similar experiments were performed.

As a result, it was confirmed that the spear of HEK293 cells can be cultured in suspension using the medium composition, and that the cells proliferate efficiently in the medium composition. In addition, it was confirmed that the culture medium composition had a smaller cell ratio and a superior cell proliferation promoting effect when cells were proliferated, compared with a culture composition containing no deacylated gellan gum. The spear cultured in the conventional medium settled on the bottom of the culture container.

The relative number of HEK293 cells, in which the number of cells cultured in the medium without deacylated gellan gum is 1, In addition, Table 27 shows the ratio of relative cell ratio, in which the ratio of cells (number of cells / number of living cells) cultured in the medium containing no deacylated gellan gum is 1.

[Table 26]

[Table 27]

Reference Example 20: Cell proliferation test by culturing insect cells

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) at 0.3% (w / v) and dissolved by stirring while heating at 90 캜. This aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. Using this solution, a culture medium composition was prepared by adding deacylated gellan gum to a Sf-900 (registered trademark) III SFM medium (manufactured by Gibco) to a final concentration of 0.015% (w / v). Subsequently, Sf9 cells (from Gibco) derived from Spodoptera frugiperda were inoculated into 100,000 cells / mL of the above-mentioned medium composition to which deacylated gellan gum had been added, and 24-well flat microplates (Corning Incorporated) to 1 mL / well. The cell suspension was incubated in an incubator at 25 DEG C for 5 days and then cultured. Then, a part of the culture medium was recovered, and the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added, and the number of viable cells was measured by a hemocyte calculator (manufactured by ERMA INC.). As a control, a culture composition not containing deacylated gellan gum was prepared and a similar experiment was conducted.

As a result, it was confirmed that Sf9 cells can be uniformly cultured in a suspension state using the medium composition and proliferated in the medium composition. In addition, it was confirmed that the culture medium composition was superior to the culture medium containing no deacylated gellan gum, in promoting cell proliferation when cells were proliferated. The cell numbers of Sf9 cells after 5 days of suspension culture are shown in Table 28. < tb > < TABLE 28 >

[Table 28]

Reference Example 21: Cell proliferation test by culturing CD34-positive cells

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) at 0.3% (w / v) and dissolved by stirring while heating at 90 캜. This aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. These solutions were used to dissolve deacylated gellan gum, 20 ng / mL of thrombopoietin (WAKO) and 100 ng / mL of stem with a final concentration of 0.015% (w / v) in StemSpan SFEM medium (StemCell Technologies) And cell factor (SCF, manufactured by WAKO) was added to prepare a medium composition. Subsequently, human umbilical cord blood-derived CD34-positive cells (manufactured by Lonza) were inoculated into the above-mentioned medium composition to which 10000 cells / mL of deacylated gellan gum had been added and cultured in a 24-well flat microplate (manufactured by Corning Incorporated) Well and dispensed at 1 mL / well. The cell suspension was incubated in a CO ₂ incubator (5% CO ₂ ) for 7 days at 37 ° C. Then, a part of the culture medium was recovered, and the same amount of trypan blue staining solution (manufactured by Invitrogen Corporation) was added, and the number of viable cells was measured by a hemocyte calculator (manufactured by ERMA INC.). Three-fold volume of medium was added to the culture medium, and the mixture was centrifuged (500 G, 5 min) to sediment all cells. As a control, a culture composition not containing deacylated gellan gum was prepared and a similar experiment was conducted.

As a result, it was confirmed that the CD34-positive cells could be uniformly cultured in a suspension state using the medium composition and proliferated in the medium composition. Further, it was confirmed that the culture medium had a cell proliferation promoting effect more than that of the conventional culture medium containing no deacylated gellan gum. In addition, it was confirmed that centrifugation caused cell sedimentation and that cells could be recovered. The relative number of cells proliferated from CD34-positive cells after 7 days of suspension culture with 1 cell number cultured in a medium containing no deacylated gellan gum is shown in Table 29. [

[Table 29]

Reference Example 22: Spear forming test

(Manufactured by WAKO) containing 0.015% of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v / v) fetal calf serum in the same manner as in Experimental Example 2, ≪ / RTI > Subsequently, HepG2 cells were added at a cell concentration of 15000 cells / mL and dispensed in 1 mL into a 24-well plate (manufactured by Corning Incorporated). These plates were allowed to stand at 37 ° C for 7 days to suspend-culture, and the formation of sphere was confirmed by microscopy. Spear cells were also precipitated by centrifugation (400 G, 5 min) and washed once with PBS (5 mL). 100 μL of tryptic-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C. for 5 minutes. To the resulting cell suspension (100 μL), DMEM medium (100 μL) containing 10% (v / v) fetal calf serum was added and a trypsin blue staining solution (Invitrogen Corporation) And the number of viable cells was measured by a hemocyte calculator (manufactured by ERMA INC.). As a result, it was confirmed that HepG2 cells formed spears in the medium composition and increased to 80,800 cells / mL. The sphere of HepG2 cells formed in the medium composition is shown in Fig.

Reference Example 23: Cell suspension test using cell line-derived spear

Dituan gum (KELKO-CRETE DG, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) at a concentration of 0.3% (w / v) and dissolved with stirring while heating at 90 캜. Using this aqueous solution, a DMEM / F-12 medium composition having a final diutenance concentration of 0.1% (w / v) was prepared. Further, an aqueous solution containing 0.5% (w / v) of native-type gellan gum (KELCO GEL HT, manufactured by San-Ei Gen F.F.I., Inc.) was prepared by heating at 90 ° C. A solution of DMEM / F-12 medium (Sigma Ltd.) containing 0.05 or 0.1% (w / v) native gellan gum was prepared using an aqueous solution.

In the same manner as in Reference Example 2, a sphere of HeLa cells was prepared, several tens of spears were added to each of the prepared medium (1 mL), the mixture was kept at 37 캜 for 1 hour, Was visually observed. As a result, it was confirmed that the sphere of HeLa cells maintained a suspension state in any of the above-mentioned medium composition. Furthermore, it was confirmed that centrifugation (200 G, 5 min) of the cell suspension containing 0.1% (w / v) diatan gum yielded the sedimentation and recovery of sphere of HeLa cells.

Reference Example 24: Cell suspension test using magnetic beads having cell adhesion ability - 1

A suspension of GEM (registered trademark, Global Eukaryotic Microcarrier, GL Sciences Inc.) coated with laminin or fibronectin was dispensed in 500 μL into a 1.5 mL volume microtube (Eppendorf) and placed in a magnetic stand (TA4899N12, Manufactured by TAMAGAWA SEIKI CO., LTD.) Was used to accumulate GEM from the above-mentioned GEM suspension, and the solvent was removed. The GEM was washed twice with DMEM medium (500 μL, manufactured by WAKO) containing 10% (v / v) fetal calf serum and suspended in the medium (500 μL). This suspension was dispensed into a cell adhesion plate, Sumilon cell tight plate 24 F (manufactured by SUMITOMO BAKELITE), in a volume of 50 μL per well. Subsequently, separately prepared HepG2 cells were added to 250,000 cells / mL and the final volume was adjusted to 500 μL / well by the same medium. These cell suspensions were manually stirred and the plates were allowed to stand overnight in a CO ₂ incubator (5% CO ₂ ). After confirming the adhesion of the cells on the GEM by a microscope, the cell suspension was transferred to a 1.5 mL micro test tube (manufactured by Eppendorf), the cell-attached GEM was accumulated using the above-mentioned magnetic stand, and the supernatant was removed.

(Manufactured by Wako Pure Chemical Industries, Ltd.) containing 0.015% of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v / v) ) Composition. The culture medium containing no such culture medium or deacylated gellan gum was added to 1 mL of the HepG2 cell-attached GEM (laminin or fibronectin coating) prepared above, suspended, and transferred to a Sumilon cell tight plate 24 F . Subsequently, the plate was allowed to stand in a CO ₂ incubator (5% CO ₂ ) for 6 days, the cell culture medium was transferred to a 1.5 mL micro test tube (manufactured by Eppendorf) and lightly pipetted on the above- - The attached GEM was pooled and the supernatant was removed. GEM was washed once with PBS (1 mL), 200 μL of tryptic-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and the mixture was incubated at 37 ° C. for 10 minutes. 800 μL of a DMEM medium containing 10% (v / v) fetal serum was added to 200 μL of the cell suspension thus obtained. An equal amount of tryptic blue staining solution (manufactured by Invitrogen Corporation) was added to a part of the cell suspension, Manufactured by ERMA INC.).

As a result, it was confirmed that GEM adhered with HepG2 cells could be cultured in a suspension state using the culture medium composition, and proliferated efficiently in the culture medium composition. Further, it was confirmed that the culture medium composition exhibited better cell proliferation promoting effect than that of the conventional culture medium containing no deacylated gellan gum. In addition, it was confirmed that HepG2 cell-attached GEM could be collected from the culture composition using magnetic force, and HepG2 cells could be recovered from such GEM.

Table 30 shows the number of HepG2 cells when cultured on GEM for 6 days in a deacylated gellan gum-containing or non-containing medium. Further, the suspension state of HepG2 cell-attached laminin-coated GEM when cultured in the medium composition is shown in Fig.

[Table 30]

Reference Example 25: Cell suspension test using magnetic beads having cell adhesion ability - 2

In the same manner as in Experimental Example 24, Fibronectin-coated GEM (registered trademark, Global Eukaryotic Microcarrier, manufactured by GL Sciences Inc.) was cultured in MF-Medium (R) mesenchymal stem cell growth medium (TOYOBO CO. LTD.). This suspension was suspended in a cell adhesion plate, Sumilon cell tight plate 24 F (manufactured by SUMITOMO BAKELITE), in a volume of 50 μL per well. Subsequently, separately prepared human bone marrow-derived mesenchymal stem cells (manufactured by Cell Applications) were added at 250,000 cells / mL, and these plates were incubated overnight in a CO ₂ incubator (5% CO ₂ ), And GEM in which mesenchymal stem cells were adhered was prepared.

(Registered trademark) mesenchymal stem cell growth medium (manufactured by TOYOBO Co., Ltd.) containing 0.015% of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) CO., LTD.) Composition was prepared. The medium containing no such medium composition or deacylated gellan gum was added to each of the prepared mesenchymal stem cell-attached GEM (fibronectin-coated) as 1 mL, suspended therein, and suspended in Sumilon cell tight plate 24 F I moved. Subsequently, the plate was allowed to stand in a CO ₂ incubator (5% CO ₂ ) for 4 days, the cell culture liquid was transferred to a 1.5 mL micro test tube (manufactured by Eppendorf) and lightly pipetted on the above- - Attached GEM was accumulated and supernatant was removed. GEM was washed once with PBS (1 mL), 200 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added, and incubated at 37 ° C. for 10 minutes. 800 μL of a DMEM medium containing 10% (v / v) fetal serum was added to 200 μL of the cell suspension thus obtained. An equal amount of tryptic blue staining solution (manufactured by Invitrogen Corporation) was added to a part of the cell suspension, Manufactured by ERMA INC.).

As a result, it was confirmed that GEM adhered with mesenchymal stem cells could be cultured in a suspension state using the culture medium composition and efficiently proliferated in the medium composition. In addition, it was confirmed that the culture medium composition exhibited better promoting effect on cell proliferation than that of the conventional culture medium containing no deacylated gellan gum. It was also confirmed that the mesenchymal stem cell-attached GEM could be collected from the medium composition using the magnetic force, and the mesenchymal stem cells could be recovered from such GEM.

Cell numbers of mesenchymal stem cells when cultured for 4 days on GEM in a deacylated gellan gum-containing or -free medium are shown in Table 31.

[Table 31]

Reference Example 26: Cell suspension test using alginate beads

The following tests were carried out according to the method of an alginate three-dimensional culture kit manufactured by PG Research. Separately prepared HepG2 cells were added to a sodium alginate solution (2.5 mL, manufactured by PG Research) at 400,000 cells / mL, and human recombinant laminin 511 (manufactured by Veritas Ltd.) was further added at 5 μg / mL, . The cell suspension was recovered with a 5 mL syringe (manufactured by TERUMO CORPORATION) having a gavage needle, and a 22 G injection needle (manufactured by TERUMO CORPORATION) was attached to the syringe. Subsequently, for each well of a 24-well plain microplate (PG Research) to which 2 mL of an aqueous calcium chloride solution (manufactured by PG Research) was added, a cell suspension was added in 10 drops. The mixture was allowed to stand at room temperature for 10 minutes, the formation of alginate beads was confirmed, the calcium chloride solution was removed, PBS (2 mL) was added, and the mixture was allowed to stand at room temperature for 15 minutes. In addition, PBS was removed, DMEM medium (WAKO, 2 mL) containing 10% (v / v) fetal serum was added, and the mixture was allowed to stand at room temperature for 15 minutes. The medium was removed, and 0.03% of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v / v) fetal serum prepared by the method similar to Reference Experiment 2 (WAKO) or deacylated gellan gum was added to each well in an amount of 1 mL and the cells were cultured in a CO ₂ incubator (5% CO ₂ ) for 8 days. The medium was changed on the fourth day of culture.

The incubated alginate beads were transferred to a 1.5 mL micro test tube (manufactured by Eppendorf) using a 1 mL tip, sodium citrate solution (1 mL, manufactured by PG Research) was added to each tube, and the mixture was incubated at room temperature for 15 Lt; / RTI > for 1 minute to dissolve alginic acid beads. Subsequently, the cells were sedimented by centrifugation at 300 G for 3 minutes, and the supernatant was removed. 200 [mu] L of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the cells, and the mixture was incubated at 37 DEG C for 5 minutes. 800 μL of a DMEM medium containing 10% (v / v) fetal serum was added to the resulting cell suspension (200 μL), and a trypsin blue staining solution (manufactured by Invitrogen Corporation) was added in an equivalent amount to a part of the cell suspension. The number of viable cells was measured by a calculator (manufactured by ERMA INC.).

As a result, it was confirmed that the alginate beads containing the HepG2 cells could be cultured in suspension using the culture medium composition, and proliferated efficiently in the culture medium composition. Further, it was confirmed that the culture medium composition exhibited better cell proliferation promoting effect than that of the conventional culture medium containing no deacylated gellan gum.

The cell numbers of HepG2 cells when cultured for 8 days in alginate beads in the deacylated gellan gum-containing or -free medium are shown in Table 32. [ In addition, the suspension state when the HepG2 cell -formed alginate beads were cultured in the culture medium composition is shown in Fig.

[Table 32]

Reference Example 27: Cell suspension test using collagen gel capsules

A: Tissue culture collagen Cell matrix (registered trademark) Type IA (Cell Matrix, manufactured by Nitta Gelatin Inc.) B: 10-fold concentration DMEM / F-12 medium (Aldrich) C: Reconstitution buffer To a solution of sodium hydroxide (100 mL) was added sodium bicarbonate (2.2 g), HEPES (4- (2-hydroxyethyl) -1-piperazinethanesulfonic acid) (4.77 g) ) Were mixed in a ratio of A: B: C = 8: 1: 1 while cooling in ice. Further, human recombinant laminin 511 (manufactured by Veritas Ltd.) was added at 5 μg / mL to prepare a collagen mixture solution (500 μL). Separately prepared HepG2 cells were added to the mixed solution at 200,000 cells / mL, and the total amount was recovered using a 1.5 mL syringe (manufactured by TERUMO CORPORATION) equipped with a 25G injection needle (manufactured by TERUMO CORPORATION). (Manufactured by BM Equipment Co., Ltd.) containing DMEM medium (WAKO) (10 mL) previously incubated at 37 占 폚 and containing 10% (v / v) The cell suspension was added dropwise in one drop using a syringe. The mixture was incubated in a water bath at 37 DEG C for 10 minutes to confirm the formation of amorphous collagen gel capsules having a diameter of about 2 mm and the same procedure as in Experimental Example 2 was carried out to obtain deacylated gellan gum (KELCOGEL CG -LA, manufactured by SANSHO Co., Ltd.) was added, and the capsules were suspended by gently stirring. Subsequently, the tubes were incubated in CO ₂ incubator (5% CO ₂ ) for 5 days.

PBS (25 mL) was added to the culture medium containing the collagen gel capsules, the collagen gel capsules were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. Again, PBS (25 mL) was added, the mixture was centrifuged, and the supernatant was removed to make the remaining volume 5 mL. To this solution was added 1% (W / V) collagenase L (20 μL, manufactured by Nitta Gelatin Inc.) and the mixture was shaken at 37 ° C for 2 hours. After confirming the dissolution of the collagen gel, PBS (10 mL) was added, the cells were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. 1 mL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the cells, followed by incubation at 37 DEG C for 5 minutes. To the resulting cell suspension, 4 mL of DMEM medium containing 10% (v / v) fetal calf serum was added, the cells were sedimented by centrifugation at 400 G for 5 minutes, and the supernatant was removed. The obtained cells were suspended in 2 mL of the same medium, and a part thereof was added with the same amount of tryptic blue staining solution (manufactured by Invitrogen Corporation), and the number of viable cells was measured with a hemocytometer (manufactured by ERMA INC.).

As a result, it was confirmed that collagen gel capsules in which HepG2 cells were embedded could be cultured in a suspension state using the culture medium composition, and cells proliferated efficiently in the culture medium composition. Further, it was confirmed that the culture medium composition exhibited better cell proliferation promoting effect than that of the conventional culture medium containing no deacylated gellan gum.

Table 33 shows the number of HepG2 cells when cultured in collagen gel capsules in a deacylated gellan gum-containing or non-containing medium for 5 days. 11 shows the suspension state when the HepG2 cell-embedded collagen gel capsules were cultured in the culture medium.

[Table 33]

Reference Example 28: Recovery test of a sphere using a filter

(WAKO) composition containing 0.015% of deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) and 10% (v / v) fetal serum by a method similar to that in Reference Example 2 . In addition, as the control, the same medium containing no deacylated gellan gum was prepared. HepG2 cell spears were formed by the method similar to that in Reference Example 2, and 86,000 cells were added to the prepared culture medium (1 mL), the mixture was allowed to stand at 37 DEG C for 1 hour, and the spear cell suspension was visually Observed. In addition, a cell suspension was added to a cell strainer (Becton, Dickinson and Company) having a mesh size of 40 mu m, and the sphere was captured on the filter. Subsequently, the spear was withdrawn into a 15 mL tube by flowing PBS (10 mL) from the back of the filter, and the spore was settled by centrifugation at 300 G for 5 minutes. The supernatant was removed, and 500 μL of trypsin-EDTA (ethylenediaminetetraacetic acid) solution (manufactured by WAKO) was added to the sphere and incubated at 37 ° C. for 5 minutes. DMEM medium (1 mL) containing 10% (v / v) fetal serum was added to the obtained cell suspension. An equal amount of tryptic blue staining solution (manufactured by Invitrogen Corporation) was added to a part of the cell suspension and counted in a hemocytometer (ERMA INC. Ltd.) to measure the number of live cells. As a result, it was confirmed that the sphere of HepG2 cells was maintained in suspension in the above-mentioned medium composition. In addition, a sphere suspension containing 0.015% of deacylated gellan gum was filtered to confirm that the cells of HepG2 cell sphere could be recovered at the same recovery rate as that of the medium containing no deacylated gellan gum. Table 34 shows the relative number recovered from the medium containing the deacylated gellan gum, in which the number of HepG2 cells recovered by the filter was 1, using a medium containing no deacylated gellan gum.

[Table 34]

Reference Example 29: Cell suspension test of sphere using various polysaccharide combinations

(Duck alginic acid NSPM, manufactured by FOOD CHEMIFA Co., Ltd.), locust bean gum (GENUGUM RL (trade name), manufactured by Nippon Kayaku Co., Ltd.) (GENUGEL WR-80-J, manufactured by SANSHO Co., Ltd.), pectin (GENUEL pectin LM-102, manufactured by SANSHO Co., Ltd.), methylcellulose (cP400, manufactured by WAKO) (KELCO CRETE DG-F, manufactured by SANSHO Co., Ltd.) and deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) To prepare a DMEM / F-12 medium composition. Spheres of HepG2 cells were prepared in the same manner as in Reference Example 2, dozens of spears were added to each medium (1 mL) prepared above, and the mixture was allowed to stand at 37 DEG C for 1 hour or overnight , And the suspension state of the sphere cells was visually observed. As a result, it was confirmed that the sphere of HepG2 cells maintained a suspension state in any of the above-mentioned medium composition. It was also confirmed in all media compositions that the addition of a 2-fold volume of medium and centrifugation of the cell suspension (500 G, 5 min) yielded sedimentation and recovery of HepG2 cell spores. After one night, the dispersion state of the spear was confirmed by visual observation, where the suspended and dispersed state was?, Some precipitated / dispersed state was?, And the precipitated state was x. The evaluation results are shown in Tables 35 and 36. In the table, - indicates unrevised.

[Table 35]

[Table 36]

Comparison of dispersibility of beads and cells - 1

The dispersed state of dextranide Cytodex (registered trademark) 1 (manufactured by GE Healthcare Life Sciences) and HeLa cell sphere was compared between the deacylated gellan gum containing medium and the methyl cellulose-containing medium prepared in the above (Comparative Example) . The results are shown in Tables (Tables 37 and 38). The dispersion state of Cytodex1 and HeLa cell spores is well correlated, and Cytodex1 can be used as a cell sphere model.

[Table 37]

[Table 38]

Comparison of dispersibility of beads and cells - 2

The dispersed state of the polystyrene beads (size 500-600 占 퐉, manufactured by Polysciences Inc.) and the HepG2 cell sphere were compared between the polysaccharide prepared in Reference Example 9 and the deacylated gellan gum-containing medium. In the evaluation, the suspended and dispersed state is?, Some precipitated / dispersed state is?, And the precipitated state is 占. The results are shown in the table (Table 39). The polystyrene beads and the dispersed state of the HepG2 cell sphere are well correlated, and the polystyrene beads can be used as a cell sphere model.

[Table 39]

Reference Example 30: Suspension culture test of rice-derived flora united tissues

Fifty seeds of Nipponbare fully ripe seeds (purchased from Koto agricultural cooperatives) selected with the salt solution were transferred to a 50 mL polystyrene tube (BD Falcon), washed with sterile water (50 mL), washed with 70% Ethanol < / RTI > water (30 mL) was stirred for 1 minute. The ethanol water was removed, and Kitchen Haiter (manufactured by Kao Corporation, 30 mL) was added and the mixture was stirred for 1 hour. Kitchen Haiter was removed and washed 4 times with sterile water (50 mL). The sterilized seeds were seeded on a Murashige Skoog based medium (M9274, Sigma Aldrich) containing 2 쨉 g / mL of 2,4-dichlorophenoxyacetic acid (Sigma Aldrich) and agar at a flow rate of 1.5 mL / Well (24-well flat microplate (manufactured by Corning Incorporated)). The cells were cultured for 3 weeks under the conditions of a dark place at 8O < 0 > C and a dark place at 30 [deg.] C, and pale yellow union tissues (1-2 mm) grown in seed cysts were harvested.

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) at 0.3% (w / v) and dissolved with stirring while heating at 90 캜. This aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. Using this solution, a final concentration of 0.03% (w / v) was added to a Murakasej Skug base medium (M9274, manufactured by Sigma Aldrich) containing 2 쨉 g / mL of 2,4-dichlorophenoxyacetic acid (manufactured by Sigma Aldrich) ) Was added with deacylated gellan gum to prepare a culture medium composition. Fifteen union tissues prepared above were added to these compositions in a 10 mL / well tube (manufactured by BM Equipment Co., Ltd.), and shake culture was performed at 25 캜 for 7 days. As a result, the rice-derived union tissue can be cultured in a suspension state using the medium composition, and the union tissue is retained in the medium composition. The suspension state when the rice-derived union tissue was cultured in the medium composition is shown in Fig.

[Preparation Example 1: Preparation of crystalline cellulose-derived cellulose nanofiber]

A commercially available crystalline cellulose (PH-101, manufactured by Asahi Kasei Chemicals Corporation) (4 parts by mass) was dispersed in pure water (396 parts by mass), and a high pressure pulverizer (Star Burstsystem) To obtain an aqueous dispersion (MNC) of cellulose nanofibers derived from crystalline cellulose. The obtained dispersion was measured by a balance in a petri dish and dried at 110 DEG C for 1 hour to remove moisture. The amount of the residue was measured, and the concentration was measured. As a result, the cellulose concentration (solid content concentration) in the water was 1.0% by mass. This aqueous dispersion was sterilized by treating in an autoclave at 121 占 폚 for 20 minutes.

[Production Example 2: Production of pulp-derived cellulose nanofiber]

A commercially available kraft pulp (LBKP, manufactured by Oji F-Tex Co., Ltd., solid content: 89 mass%) (5 mass parts) was dispersed in pure water (145 mass parts), and MASUKO SANGYO CO., LTD. The pulverized slurry was pulverized 9 times at 1500 rpm by using a millstone grinder (Masscolloider) manufactured by the same manufacturer. The above-mentioned pulp slurry was treated at 220 MPa for 300 times using a high-pressure grinding apparatus (Star Burstsystem) manufactured by Sugino Machine Limited to obtain an aqueous dispersion (PNC) of nanocellulose. The obtained dispersion was measured by a balance in a petri dish and dried at 110 DEG C for 1 hour to remove moisture. The amount of the residue was measured, and the concentration was measured. As a result, the cellulose concentration (solid content concentration) in water was 1.6% by mass. This aqueous dispersion was sterilized by treating in an autoclave at 121 占 폚 for 20 minutes.

[Preparation Example 3: Preparation of chitin nanofiber]

20 parts by mass of commercially available chitin powder (manufactured by KOYO CHEMICAL CO., LTD.) Was dispersed in pure water (980 parts by mass), and a high pressure grinding apparatus (Star Burstsystem, And an aqueous dispersion (CT) of chitin nanofibers was obtained. The obtained dispersion was measured by a balance in a petri dish and dried at 110 DEG C for 1 hour to remove moisture. The amount of the residue was measured, and the concentration was measured. As a result, the chitin concentration (solid content concentration) in water was 2.0 mass%. This aqueous dispersion was sterilized by treating in an autoclave at 121 占 폚 for 20 minutes.

[Test Example 1: Measurement of average fiber diameter D and average fiber length L]

The average fiber diameter (D) of the nanofibers was measured as follows. First, Okenshoji Co., Ltd. The collodion support membrane manufactured by JEOL Ltd. Was applied to the hydrophilic treatment for 3 minutes with a commercially available ion creaner (JIC-410). Several droplets of the nanofiber dispersion (dilution with ultrapure water) prepared in Production Examples 1 to 3 were added dropwise and dried at room temperature. Hitachi, Ltd. (TEM, H-8000) (10,000 times) at an acceleration voltage of 200 kV. Using the obtained images, the fiber diameter of each of nanofibers (number of specimens: 200 to 250) was measured, and the average value thereof was defined as the average fiber diameter (D).

The average fiber length (L) was obtained by diluting the nanofiber dispersion prepared in Production Example with pure water to 100 ppm and uniformly dispersing the nanofibers using an ultrasonic washing machine. This nanofiber dispersion liquid was previously cast on a silicon wafer subjected to hydrophilization treatment using concentrated sulfuric acid and dried at 110 DEG C for 1 hour to be used as a sample. The length of each fiber of each nanofiber (sample number: 150 to 250) was measured using the image obtained by observing the obtained sample with a scanning electron microscope (SEM, JSM-7400 F) (2,000 times) Was taken as the average fiber length (L).

The average fiber diameter D and the average fiber length L of the nanofibers obtained in Production Examples 1 to 3 were measured, and the aspect ratio L / D was measured from these values. Table 40 shows the obtained results.

[Table 40]

[Examples 1 to 4]

Using the nanofiber dispersions and the deacylated gellan gum aqueous solutions prepared in the above-mentioned Production Examples 1 to 3, the culture compositions described in the following Table 41 were prepared.

First, sterilized water was added to the cellulose nanofibers MNC, PNC and chitin nanofibers prepared in Production Examples 1 to 3, and each was diluted with a 1% (w / v) aqueous dispersion. On the other hand, 99 parts of sterilized water of skin was added to deacylated gellan gum (KELCOGELCG-LA, manufactured by SANSHO Co., Ltd.: DAG) (1 part by mass) and dissolved and sterilized by autoclaving at 121 DEG C for 20 minutes , And 1% (w / v) aqueous solution of deacylated gellan gum.

The aforementioned 1% (w / v) dispersion or aqueous solution (1 part skin) was placed in a 50 mL conical tube, sterilized water of 49 parts skin was added and the mixture was pipetted until uniform. After filtration by sterilization with a 0.22 mu m filter, 50 parts of DMEM (high glucose, manufactured by Aldrich, a predetermined amount of sodium hydrogencarbonate) was added to the mixture in a concentration of 2 times, and the mixture was mixed by pipetting, A medium composition with a fiber concentration of 0.01% (w / v) was prepared.

Similarly, a medium composition was prepared by adding a nanofiber dispersion or a deacylated gellan gum aqueous solution such that the final desired concentration was 0.01 to 0.1% (w / v).

[Example 5 and Comparative Examples 2 to 5]

(Example 5), locust bean gum (GENUGUM RL-200-J, manufactured by SANSHO Co., Ltd.: LBG) (manufactured by SANSHO Co., Ltd., 2), xanthan gum (KELTROL CG, Xan manufactured by SANSHO Co., Ltd.) (Comparative Example 3), dianthum gum (KELCO CRETE DG-F, manufactured by SANSHO Co., , 99 parts by mass of sterilized water was added to 1 part by mass of alginic acid Na (Duc alginic acid NSPM, manufactured by Food Chemifa Co., Ltd., Alg) (Comparative Example 5) and autoclaved at 121 占 폚 for 20 minutes. And sterilized.

The polysaccharide solution prepared as described above was applied to operations similar to those of Examples 1 to 4 to prepare a medium composition to which the polysaccharide solution was added such that the final concentrations were 0.03, 0.05, 0.07, and 0.1% (w / v) did.

[Test Example 2: Evaluation of suspension action - 1]

Polystyrene beads (200-300 占 퐉, manufactured by Polysciences Inc.) were added to the medium compositions of Examples 1 to 5 and Comparative Examples 2 to 5 and uniformly dispersed beads in the medium composition by vortex stirring After the confirmation, the composition was allowed to stand at room temperature (25 캜) for one day to confirm the dispersion state of the beads by visual observation. A state in which the beads were uniformly suspended in the medium composition was evaluated as?, A state in which the supernatant was partially caused by?, And a state of precipitation was evaluated as?. The results are shown in Table 41.

As a result, the medium compositions of Examples 1 to 4 showed the action of suspending the beads. In Example 5, the bead suspension activity was shown at room temperature, but the bead was sedimented by heating at 37 占 폚, and no suspension effect was obtained under cell culture conditions. In Comparative Examples 2 to 5, the beads completely settled on the bottom surface.

[Table 41]

* The κ-carrageenan (Car) of Example 5 exhibited a suspension action at 25 ° C., whereas at 37 ° C. equivalent to cell culture conditions, the suspension was lost on the spot, and the beads precipitated. For the other media, the same results were obtained at 37 ° C and 25 ° C.

[Test Example 3: Evaluation of suspension action - 2]

In the same manner as in Test Example 2, the suspension compositions in the low concentration range (0.01 to 0.04% (w / v)) of the medium compositions of Examples 2, 4 and 5 and Comparative Example 2 were evaluated in detail. Polystyrene beads were added, and the composition was allowed to stand for 2 days, and the dispersed state of the beads was confirmed by visual observation. The suspended and dispersed states were evaluated as & cir & and the sedimentation state was evaluated as x. For some settled / dispersed conditions, the bead suspension rate was calculated based on the height of the suspended area in a 10 mL conical tube. Table 42 shows the results.

[Table 42]

* Represents the bead suspension ratio. PNC showed a suspending action at a concentration of 0.015% (w / v) or more, and the culture medium of Example 4 at a concentration of 0.015% (w / v) or more. The medium composition of Example 2 shows that as the concentration is increased, the suspension action is improved stepwise. When the medium composition of Example 5 contained 0.02% or more, the suspension exhibited a suspension action at 25 ° C, but lost the suspension effect at 37 ° C, and the beads precipitated (*). For the other media, the same results were obtained at 37 ° C and 25 ° C.

[Test Example 4: Viscosity of Medium Composition]

The viscosity of the medium compositions of Examples 1 to 5 and Comparative Examples 2 to 5 was evaluated using a sound bending vibration viscometer (SV-1 A, A & D Company Ltd.) under the condition of 25 占 폚. The results are shown in Fig. The results obtained show that the medium composition of the present invention shows no remarkable increase in viscosity as compared with the viscosity of a general medium, because the content of the nanofiber or polysaccharide thickener is very small. Comparison with the results of Test Example 2 shows that there is no correlation between the viscosity and the suspending action.

[Test Example 5: Observation of the medium composition by scanning electron microscope]

The medium composition prepared in Examples 1 to 5 and Comparative Examples 3 to 4 was cast on a silicon wafer which had been subjected to surface hydrophilization treatment by using concentrated sulfuric acid in advance and was heated at 110 ° C (Comparative Example 1 room temperature) to 1 Lt; / RTI > Pure water was poured thereon to remove excess salt content and the composition was dried again at 110 DEG C for 1 hour and used as a sample. The above-mentioned sample is supplied by JEOL Ltd. Were observed using a preparative scanning electron microscope (SEM, JSM-7400F) (10,000 times). The results of observation of the media compositions of Examples 1 to 4 and Comparative Examples 3 and 4 are shown in Figs. 14 to 21. Fig.

As a result of observation, in Examples 1 to 4 and Example 5 dried at room temperature, a large number of fibers were observed, whereas in Example 5 and Comparative Examples 3 and 4 which were dried at 110 占 폚, no fibers were observed at all. In addition, a plurality of spherical objects observed in the observed image are those in which a salt component in the medium is precipitated. From these results, it was suggested that the fiber structure contained in the medium composition could contribute to the suspension action.

[Test Example 7: Spear-suspension action]

The human liver cancer cell line HepG2 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) Was suspended at 50,000 cells / mL in a DMEM medium (WAKO) containing 10% (v / v) fetal calf serum, ) Was inoculated on EZ SPHERE (manufactured by ASAHI GLASS CO., LTD.) And cultured in a CO ₂ incubator (5% CO ₂ ) for 2 days. The spear suspension (80 mL) obtained was subjected to centrifugation treatment (800 rpm, 5 minutes) to sediment the spores, and the supernatant was removed to prepare a spear suspension (4.5 mL). Subsequently, 10 mL of the medium composition of Examples 1 to 4, Comparative Example 1 and Comparative Examples 3 to 5 was added to a 15 mL conical tube, and a sphere suspension (100 μL) of HepG2 cells was further added. Spear was dispersed by pipetting and incubated at 37 DEG C for 5 days to visually observe the dispersion state of the spear in the culture medium composition. A state in which the sphere was uniformly suspended in the medium composition was rated as?, A state in which the supernatant was generated was evaluated as?, And a sedimentation state was evaluated as?. Observation results of the medium compositions of Examples 1 to 5 and Comparative Examples 3 to 5 are shown in Table 43 and Figs.

As a result, in the medium compositions of Examples 1 to 4, a suspended state was found even after culturing for 6 days. On the other hand, in the medium compositions of Example 5 and Comparative Examples 3 to 5, all of the spores were precipitated, and the spores were aggregated with each other.

[Table 43]

[Examples 1 'to 4']

Sterilized water was added to the cellulose nanofibers MNC, PNC and chitin nanofibers prepared in Preparation Examples 1 to 3 to prepare aqueous dispersions of 1% (w / v) water, respectively. On the other hand, 99 parts of sterilized water of skin was added to deacylated gellan gum (KELCOGELCG-LA, manufactured by SANSHO Co., Ltd.: DAG) (1 part by mass) and dissolved and sterilized by autoclaving at 121 DEG C for 20 minutes , 1% (w / v) aqueous solution. Using a 1% (w / v) fiber dispersion or an aqueous solution of deacylated gellan gum prepared above, 10% (v / v) water was added so that the final concentration would be 0.01%, 0.03%, 0.06% and 0.1% (w / v) Was added to a DMEM medium (manufactured by NISSUI PHARMACEUTICAL CO., LTD., High-glucose) containing a fetal calf serum to prepare a medium composition.

[Example 5 ', and Comparative Examples 3' to 5 ']

KELCO CRETE DG-F, SANSHO Co (trade name) manufactured by SANSHO Co., Ltd .; and Xanthan gum; 99 parts by mass of sterilized water was added to 1 part by mass of alginic acid Na (Duc alginic acid NSPM, Food Chemifa Co., Ltd., manufactured by Food Chemifa Co., Ltd.) and autoclaved at 121 占 폚 for 20 minutes To prepare a 1% (w / v) polysaccharide aqueous solution, respectively. Each polysaccharide aqueous solution prepared as described above was applied to an operation similar to that of Examples 5 to 8 to a DMEM medium (manufactured by NISSUI PHARMACEUTICAL CO., LTD.) Containing 10% (v / v) A culture medium composition was prepared by adding a polysaccharide aqueous solution to a final concentration of 0.01%, 0.03%, 0.06%, and 0.1% (w / v).

[Test Example 8: Cell proliferation test]

Example 1 'to Example 5' and Comparative Example 3 were performed so that the human breast cancer cell line MCF-7 (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) And human melanoma cell line A375 (ATCC) To the culture composition prepared in Comparative Example 5 ', and dispensed in a well of a 96-well, ultra-low-adhesion surface microplate (Corning Incorporated, # 3474) so as to be 150 μL per well. As a negative control, MCF7 cells or A375 cells were suspended in the medium containing no nanofibers or polysaccharides, and the suspension was dispensed. Subsequently, this plate was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for up to 6 days in a stationary state. 150 μL of ATP reagent (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added and suspended in the culture medium after 2 days and 6 days of culture, and the medium was left at room temperature for about 10 minutes. The emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the number of living cells was measured by subtracting the emission value of the medium alone.

As a result, it was confirmed that the culture medium containing PNC, MNC, or nanochitin could be cultured in a uniformly dispersed state without excessive size of cell aggregation masses, and proliferated efficiently. On the other hand, in the culture medium composition containing sodium alginate, no promotion of proliferation was observed. The RLU values (ATP measurement, luminescence intensity) of the MCF7 cells after 2 days and 6 days after the stationary culture are shown in Tables 44 to 47 and RLU values after 6 days are shown in Figs. 30 to 33, and the results of A375 cells are shown in Tables 48 to 49 The RLU values after 51 and 6 days are shown in Figs. The results of MCF7 cells and A375 cells are shown in Fig. 38 and Fig. 39, respectively, in the observation of the agglutination mass of the culture for 2 days.

[Table 44]

[Table 45]

[Table 46]

[Table 47]

[Table 48]

[Table 49]

[Table 50]

[Table 51]

[Test Example 9: Conservation test using 3T3-L1 cells]

The mouse adipose precursor cell line 3T3-L1 (manufactured by ATCC) was inoculated on a 10 cm polystyrene petri dish using 10% FBS-containing DMEM medium and cultured in an incubator set at 5% CO ₂ , 37 ° C. When the 3T3-L1 cells became confluent, the medium was aspirated, the FBS was removed by D-PBS (Wako Pure Chemical Industries, Ltd.), and a solution containing 0.25% trypsin and 1 mM EDTA 1 ml) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the above-mentioned polystyrene petri dish. After confirming the detachment of the cells, DMEM medium containing 10% FBS was added, cells were recovered from the Petri dish, and transferred to a centrifuge tube. The cells were centrifuged at 300 x g and the supernatant was removed. Prepare cell suspensions of about 100 × 10 ⁴ cells / mL, add 100 μL of cell suspension in 1.5 mL microtube, and prepare the cells in the same manner as in Example 1 and Example 1, which had been previously prepared to contain 10% (v / v) FBS 100 μL of each of the medium compositions of Example 2, Example 4, and Example 5, and Comparative Example 3 and Comparative Example 5 were added, and the mixture was pipetted to prepare a cell suspension.

The cell suspension was kept in a closed state at room temperature for 10 days, and after 3 or 10 days, a portion of the cell suspension was diluted 1/10 with DMEM medium containing 10% FBS. ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to and suspended in the diluted cell suspension (100 μL), and the medium was allowed to stand at room temperature for 15 minutes. The emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the number of living cells was measured by subtracting the emission value of the medium alone. The negative control was a sample containing medium alone without polysaccharide.

As a result, as for the cell viability of each of the negative control compositions or the media compositions of Comparative Example 3 and Comparative Example 5, the ATP value was remarkably decreased at room temperature storage for 3 to 10 days, and in Examples 1 to 2 and In the medium composition of Example 4, the decrease in ATP value was suppressed, and thus the cytoprotective effect was exhibited. Table 52 shows the results of the number of living cells.

[Table 52]

[Test Example 10: Conservation test using CHO-K1 cells]

Chinese hamster I shochu CHO-K1-hIFNβ cells (Kitakyushu National College of Technology in, provided by Professor Kawahara) a, 10% FBS-containing using F12 medium, and seeded on a 10 cm polystyrene petri dishes, 5% CO _2, And incubated in an incubator set at 37 ° C. When the CHO-K1-hIFN [beta] cells become confluent, the medium is aspirated, the FBS is removed by D-PBS (Wako Pure Chemical Industries, Ltd.), and 0.25% trypsin and 1 mM EDTA Solution (1 ml) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the above-mentioned polystyrene petri dish. After confirming cell detachment, F12 medium containing 10% FBS was added, cells were recovered from the Petri dish, and transferred to a centrifuge tube. The cells were centrifuged at 300 x g and the supernatant was removed. Embodiment of about 5 × 10 ⁶ cells to prepare a cell suspension / mL, and added to the cell suspension of 25 μL in a 1.5 mL microtube, and which has been previously prepared to contain 10% (v / v) FBS Example 2 and carrying out 25 μL each of the medium composition of Example 4 was added, and the mixture was pipetted to prepare a cell suspension.

The cell suspension was kept in a closed state at room temperature for 1 day, and a portion of the cell suspension was diluted 1/10 with F12 medium containing 10% FBS. ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to and suspended in the diluted cell suspension (100 μL), and the suspension was allowed to stand at room temperature for about 10 minutes. Then, FlexStation 3 (manufactured by Molecular Devices ), And the number of live cells was measured by subtracting the luminescence value of the medium alone. The negative control was a sample containing medium alone without polysaccharide.

As a result, the cell survival rate in the negative control group was lowered by keeping the cell temperature at room temperature for 1 day; In the medium compositions of Examples 2 and 4, the ATP value at the time of seeding was shown and the cell protection effect was shown. Table 53 shows the results of the number of living cells.

[Table 53]

[Test Example 11: Conservation test using 3T3-L1 cells, change in polysaccharide concentration]

The mouse adipose precursor cell line 3T3-L1 (manufactured by ATCC) was inoculated on a 10 cm polystyrene petri dish using 10% FBS-containing DMEM medium and cultured in an incubator set at 5% CO ₂ , 37 ° C. When 3T3-L1 cells became 40% confluent, the medium was aspirated off, FBS was removed by D-PBS (Wako Pure Chemical Industries, Ltd.), and 0.25% trypsin and 1 mM EDTA Solution (1 ml) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the above-mentioned polystyrene petri dish. After confirming the detachment of the cells, DMEM medium containing 10% FBS was added, cells were recovered from the Petri dish, and transferred to a centrifuge tube. The cells were centrifuged at 300 x g and the supernatant was removed. A cell suspension of about 100 x 10 ⁴ cells / mL was prepared, 100 μL of the cell suspension was added to a 1.5 mL microtube, and the cells were prepared in the same manner as in Example 2 and Example 2, which had been previously prepared to contain 10% (v / v) FBS 100 μL each of the medium compositions of Example 4 and Comparative Example 5 were added and the mixture was pipetted to prepare a cell suspension.

The cell suspension was kept in a closed state at room temperature for 8 days, and after a lapse of 0 day, 5 days, or 8 days, a part of the cell suspension was diluted 1/5 with DMEM medium containing 10% FBS. ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to and suspended in the diluted cell suspension (100 μL), and the medium was allowed to stand at room temperature for 15 minutes. The emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the number of living cells was measured by subtracting the emission value of the medium alone. The negative control was a sample containing medium alone without polysaccharide.

As a result, as for the cell viability of the negative control or the medium composition of Comparative Example 3, the ATP value was remarkably decreased at room temperature storage for 5 to 8 days, and in the medium compositions of Examples 2 and 4, ATP The decrease in the value was suppressed, thus showing a cytoprotective effect. Table 54 shows the results of the number of living cells.

[Table 54]

[Test Example 12: Effect on cell survival action on MDCK cells]

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) so as to be 0.3% (w / v) and dissolved by stirring at 90 ° C. The aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. Using this solution, a final concentration of 0.005% (w / v) or 0.015% of deacylated gellan gum was added to EMEM medium (Wako Pure Chemical Industries, Ltd.) containing 10% (v / v) And a EMEM medium without deacylated gellan gum were prepared. Subsequently, MDCK (manufactured by DS PHARMA BIOMEDICAL CO., LTD.), Which had been cultured in a medium containing no serum for 1 day (fasting treatment), was inoculated into 100,000 cells / mL of deacylated gellan gum , And dispensed in a well of a 96-well, ultra-low-adhesion surface microplate (Corning Incorporated, # 3474) so as to have a volume of 100 μL per well. Each plate was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 15 days in a stationary state. After incubation for 2, 6, 9, 12 and 15 days, ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to the culture medium to calculate a suspension. And the light emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices), and the number of living cells was measured by subtracting the emission value of the medium alone.

As a result, it has become clear that, by culturing the MDCK cells on a low adhesion plate using the medium composition of the present invention, the decrease in the number of living cells can be suppressed. Table 55 shows RLU values (ATP measurement, luminescence intensity) in each culture.

[Table 55]

[Test Example 13: Effect on cell survival effect on Vero cells]

The deacylated gellan gum (KELCOGEL CG-LA, manufactured by SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) so as to be 0.3% (w / v) and dissolved by stirring at 90 ° C. The aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. Using this solution, a medium composition was prepared by adding 0.005% (w / v) or 0.015% of deacylated gellan gum to an Emedium199 medium (Sigma Ltd.) containing 5% (v / v) An undiluted medium composition without deacylated gellan gum was prepared. Subsequently, dehydrated gellan gum was added to 100,000 cells / mL of the monkey kidney skin cell line Vero (manufactured by DS PHARMA BIOMEDICAL CO., LTD.) Cultured for 1 day in a medium containing no serum The above-mentioned medium composition was inoculated and dispensed in a well of a 96-well, ultra-low-adhesion surface microplate (Corning Incorporated, # 3474) so as to have a volume of 100 μL per well. Each plate was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 15 days in a stationary state. After incubation for 2, 6, 9, 12 and 15 days, ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to the culture medium to calculate a suspension. And the light emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices), and the number of living cells was measured by subtracting the emission value of the medium alone.

As a result, it has become clear that, by culturing the Vero cells on a low adhesion plate using the medium composition of the present invention, the reduction in the number of living cells can be suppressed. Table 56 shows RLU values (ATP measurement, luminescence intensity) in each culture.

[Table 56]

[Test Example 14: Effect of each substrate on MDCK cell proliferating action]

(PNC), chitin nanofibers (Biomass nanofiber BiNFi-S 2 mass%, Sugino Machine Limited) and deacylated gellan gum (KELCOGEL CG-LA, SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) so as to be 1% (w / v) and dissolved by stirring at 90 ° C. The aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. (W / v), 0.03%, or 0.1% cellulosic nanofibers in a final concentration of 0.01% (w / v) in a serum-free medium KBM220 medium (manufactured by KOHJIN BIO) (w / v), 0.015%, 0.03%, 0.06%, or a final concentration of 0.005% (w / v) in a serum-free medium KBM220 medium (manufactured by KOHJIN BIO) 0.1% of deacylated gellan gum was added and the above-mentioned unmodified medium composition containing no substrate was prepared. Subsequently, MDCK (manufactured by DS PHARMA BIOMEDICAL CO., LTD.), Which was cultured in a medium containing no serum for 1 day (fasting treatment), was added to 100,000 cells / mL of each substrate , And dispensed in a well of a 96-well, ultra-low-adhesion surface microplate (Corning Incorporated, # 3474) so as to have a volume of 100 μL per well. Each plate was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 14 days in a standing state. After culturing for 3, 7, 10, and 14 days, ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to the culture medium to give a suspension. The suspension was allowed to stand at room temperature for about 10 minutes, The emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the number of living cells was measured by subtracting the emission value of the medium alone.

As a result, when MDCK cells were cultured on a low adhesion plate using deacylated gellan gum, nano-cellulose fiber PNC, or chitin nanofiber, which is a culture medium composition of the present invention, the proliferation promoting action on MDCK cells was inhibited Lt; / RTI > Among them, chitin nanofiber showed the strongest effect. Table 57 shows RLU values (ATP measurement, luminescence intensity) in each culture.

[Table 57]

[Test Example 15: Effect of chitin nanofiber on MDCK proliferation action]

Cellulose nanofibers (PNC), chitin nanofibers (biomass nanofibers BiNFi-S 2 mass%, Sugino Machine Limited) and deacylated gellan gum (KELCOGEL CG-LA, SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) so as to be 1% (w / v) and dissolved by stirring at 90 ° C. The aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. A culture medium in which chitin nanofibers at 0.0001% (w / v), 0.0003%, 0.001%, 0.003%, 0.01%, 0.02%, or 0.03% of chitin nanofibers were added to a serum-free medium KBM220 medium (manufactured by KOHJIN BIO) The culture medium prepared by adding 0.005% (w / v), 0.015%, or 0.03% of deacylated gellan gum to a serum medium KBM220 medium (manufactured by KOHJIN BIO) and the above-mentioned unmodified medium composition containing no substrate were prepared did. Subsequently, MDCK (manufactured by DS PHARMA BIOMEDICAL CO., LTD.), Which had been cultured in a medium containing no serum for 1 day (fasting treatment), was soaked in 100,000 cells / mL of the above deacylated Gellan gum or chitin nanofibers, and dispensed in a well of a 96-well, ultra-low-adhesion surface microplate (Corning Incorporated, # 3474) to 100 μL per well. Each plate was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 14 days in a standing state. After culturing for 5, 9, 12 and 15 days, ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to the culture medium to give a suspension. The suspension was allowed to stand at room temperature for about 10 minutes, The light emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the emission value of the medium alone was subtracted to measure the number of living cells as an average value of three points.

As a result, when the MDCK cells were cultured on the low adhesion plates using the deacylated gellan gum and chitin nanofibers as the culture medium of the present invention, the proliferation promoting action on the MDCK cells was observed in the two compositions containing the respective substrates . Among them, the chitin nanofiber exhibited the effect of promoting the growth at a concentration of 0.0001% or more, particularly 0.001% or more. Table 58 shows RLU values (ATP measurement, luminescence intensity) in each culture.

[Table 58]

[Test Example 16: Effect of chitin nanofiber on MDCK cell proliferating action]

Primary culture;

(Milli-Bi-Si) nanoparticles prepared in Preparation Example 2 or 1% (w / v) of chitin nanofibers (biomass nanofibers BiNFi-S 2 mass%, Sugino Machine Limited) Q water) and dissolved by stirring with heating at 90 占 폚. The aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. A culture medium in which chitin nanofibers with a final concentration of 0.01% (w / v) were added to a serum-free medium KBM220 medium (manufactured by KOHJIN BIO), and a culture medium containing no chitin nanofibers KBM220 medium (KOHJIN BIO) An addition medium composition was prepared. Subsequently, MDCK (manufactured by DS PHARMA BIOMEDICAL CO., LTD.), Which had been cultured in a medium containing no serum for 1 day (fasting treatment), was cultured in the same manner as in the above-mentioned chitin The above-mentioned medium composition with nanofibers added was inoculated and dispensed in a 125 ml Erlenmeyer flask (Corning Incorporated, # 431405) to one 30 mL per flask. The flask was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 6 days in a stationary state. On the 0th and 6th days, the culture medium was suspended in a pipette, and 100 μL was distributed at 3 points. An ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) , And this was allowed to stand at room temperature for about 10 minutes. The light emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the number of living cells was measured by subtracting the emission value of the medium alone.

Subculture;

To confirm the effect on subculture, a cell suspension obtained by culturing MDCK cells for 6 days in a medium containing 0.01% chitin nanofibers was studied. The cell suspension obtained by mixing the cell suspension (3 ml) and the undiluted medium composition (27 ml) to obtain the chitin nanofiber concentration at 0.001%, and the cell suspension (3 ml) and chitin at a final concentration of 0.01% (w / v) The cell suspension obtained by mixing the culture medium (27 ml) containing the nanofibers with the chitin nanofiber concentration of 0.01% was added to each 125 ml Erlenmeyer flask (Corning Incorporated, # 431405) 30 mL. The flask was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 14 days in a stationary state. On the 0th, 7th and 14th days, the culture medium was suspended in a pipette, 100 μL was distributed at 3 points, and 100 μL of ATP reagent (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the emission value of the medium alone was subtracted to obtain the number of living cells as an average value of three points Respectively.

As a result, the MDCK cells were cultured in the Erlenmeyer flask using chitin nanofibers as the culture medium of the present invention, and the proliferation promoting action on MDCK cells was observed. In the case of adding a medium containing chitin nanofibers, the proliferation of MDCK cells is observed, and it has become clear that subculture can be conveniently carried out without treatment with trypsin or the like. The RLU value (ATP measurement, luminescence intensity) in the preliminary culture is shown in Table 59, and the RLU value (ATP measurement, luminescence intensity) in the subculture is shown in Table 60. [

[Table 59]

[Table 60]

[Test Example 17: Comparison of promoting action of chitin nanofibers in MDCK cell proliferation in each medium]

Cellulose nanofibers (PNC), chitin nanofibers (biomass nanofibers BiNFi-S 2 mass%, Sugino Machine Limited) and deacylated gellan gum (KELCOGEL CG-LA, SANSHO Co., Ltd.) was suspended in ultrapure water (Milli-Q water) so as to be 1% (w / v) and dissolved by stirring at 90 ° C. The aqueous solution was sterilized in an autoclave at 121 占 폚 for 20 minutes. The culture medium was prepared by adding 0.001% (w / v) or 0.01% chitin nanofibers to a serum-free medium KBM220 medium (manufactured by KOHJIN BIO) or Cosmedium 012 medium (manufactured by COSMO BIO co., Ltd.) The medium composition prepared by adding 0.03% of the deacylated gellan gum to the medium KBM220 or Cosmedium 012 medium and the aforementioned unmodified medium composition not containing the above-mentioned substrate was prepared. Subsequently, MDCK (manufactured by DS PHARMA BIOMEDICAL CO., LTD.), An adult kidney tubular epithelial cell line cultured for 1 day in a serum-free medium (fasting treatment), was added to 100,000 cells / The above-mentioned medium composition added with true gellan gum or chitin nanofibers, and dispensed in a well of a 96-well, ultra-low-adhesion surface microplate (Corning Incorporated, # 3474) so as to have a volume of 100 μL per well. Each plate was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 12 days in a standing state. (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to the culture medium on days 4, 8, and 12, and the suspension was allowed to stand at room temperature for about 10 minutes. Luminescence intensity (RLU value) was measured by a spectrophotometer (manufacturer) and the emission value of the medium alone was subtracted to measure the number of living cells as an average value of three points.

As a result, MDCK cells were cultured on a low adhesion plate using deacylated gellan gum and chitin nanofibers, which were the culture media of the present invention, to promote MDCK cell proliferation by adding two substrates of deacylated gellan gum and chitin nanofibers Respectively. Among them, chitin nanofibers showed high proliferative capacity even using Cosmedium012 medium at a concentration of 0.001% or more. The cell state on the fourth day was observed under a microscope. As a result, the cell aggregation mass (spear) was dispersed only in the medium condition using the deacylated gellan gum. On the other hand, in the medium condition using the chitin nanofiber, the spear and cell were proliferated as grape clusters. The RLU value (ATP measurement, luminescence intensity) in the culture using KBM220 medium is shown in Table 61, and the RLU value (ATP measurement, luminescence intensity) in the culture using Cosmedium012 medium is shown in Table 62. [ The results of microscopic observation of the culture for 4 days are shown in Fig.

[Table 61]

[Table 62]

[Test Example 18: Comparison of MDCK cell proliferative activity of chitosan nanofiber and chitin nanofiber]

Chitosan nanofibers (biomass nanofibers BiNFi-S, 1 mass%, Sugino Machine Limited) and chitin nanofibers (biomass nanofibers BiNFi-S, 2 mass%, Sugino Machine Limited) (Milli-Q water) so as to be 1% (w / v), and stirred at 90 ° C in the same manner as in Reference Example 1 Were each autoclaved at 121 占 폚 for 20 minutes. A culture medium prepared by adding chitosan nanofibers or chitin nanofibers at a final concentration of 0.001% (w / v), 0.003%, 0.01%, or 0.03% to a serum-free medium KBM220 medium (manufactured by KOHJIN BIO), a serum-free medium KBM220 medium A culture medium to which a final concentration of 0.03% of deacylated gellan gum was added, and an undiluted medium composition not containing the above-mentioned substrate were prepared. Subsequently, MDCK (manufactured by DS PHARMA BIOMEDICAL CO., LTD.), An adult kidney tubular epithelial cell line cultured for 1 day in a serum-free medium (fasting treatment), was added to 100,000 cells / The above-mentioned medium composition with added true gellan gum, chitosan nanofibers or chitin nanofibers was inoculated and dispensed into wells of a 96-well, ultra-low adhesion surface microplate (Corning Incorporated, # 3474) to 100 μL per well did. Each plate was incubated in a CO ₂ incubator (37 ° C, 5% CO ₂ ) for 12 days in a standing state. On the 7th and 11th days, ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added to the culture medium to give a suspension. The suspension was allowed to stand at room temperature for about 10 minutes. FlexStation 3 (manufactured by Molecular Devices) (RLU value) was measured by a light source, and the emission value of the medium alone was subtracted to determine the number of living cells as an average value of three points.

As a result, when the MDCK cells were cultured on the low adhesion plate using the chitosan nanofibers and chitin nanofibers, which are the culture compositions of the present invention, higher proliferation promoting action was observed than that of the deacylated gellan gum. In addition, chitin nanofibers exhibited a high proliferative capacity even at a concentration of 0.001%, and chitosan nanofibers showed a high proliferation capacity from 0.01%. Table 63 shows RLU values (ATP measurement, luminescence intensity).

[Table 63]

[Test Example 19: Macaca fascicularis primary hepatocyte preservation test]

Cellulose nanofiber (PNC) prepared in Preparation Example 2 and 1 mass% (w / v) of κ carrageenan (GENUGEL WR-80-J, SANSHO Co., Ltd. Car) prepared in the same manner as in Example 5, Aqueous solution was used. The culture medium was prepared by adding a final concentration of 0.03% (w / v), 0.1% PNC or carrageenan to a Williams' E medium (manufactured by Life Technologies, Inc.) containing 10% FBS, To prepare a medium composition. Subsequently, fresh Macaca fascicularis primary hepatocyte (manufactured by Ina Research Inc.) was mixed with the above-mentioned medium composition to which PNC or carrageenan had been added so as to be 2,500,000 cells / mL, and Cryogenic cell freezing vial (Thermo SCIENTIFIC Priest). In addition, the above-mentioned medium containing no substrate and Macaca fascicularis primary hepatocytes were distributed. The above-mentioned operation was carried out in two lots. Subsequently, the present tube was transported while being kept in a cold state for 2 days under refrigeration (about 4 ° C). The cell suspension transported under refrigerated conditions for 2 days was measured for cell viability in suspension using Trippine Blue reagent (manufactured by Life Technologies, Inc.).

As a result, fresh survival rate of monkey primary hepatocytes was maintained by refrigerating and transporting fresh monkey primary hepatocytes by using PNC as the culture medium composition of the present invention. On the contrary, carrageenan did not show such an effect. The survival rates are shown in Table 10.

[Table 64]

[Test Example 20: Evaluation of proliferation ability after re-sowing]

The mouse adipose precursor cell line 3T3-L1 (manufactured by ATCC) was inoculated on a 10 cm polystyrene petri dish using 10% FBS-containing DMEM medium and cultured in an incubator set at 5% CO ₂ , 37 ° C. When the 3T3-L1 cells became confluent, the medium was aspirated, the FBS was removed by D-PBS (Wako Pure Chemical Industries, Ltd.), and a solution containing 0.25% trypsin and 1 mM EDTA 1 ml) (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the above-mentioned polystyrene petri dish. After confirming the detachment of the cells, DMEM medium containing 10% FBS was added, cells were recovered from the Petri dish, and transferred to a centrifuge tube. The cells were centrifuged at 300 x g and the supernatant was removed. A cell suspension of about 200 x 10 ⁴ cells / mL was prepared, and 150 μL of the cell suspension was added to a 1.5 mL microtube, and the cell suspension of Example 2 (PNC) prepared previously to contain 10% (v / v) FBS (Concentration 0.06%) to Example 4 (DAG concentration 0.03%) and Comparative Example 5 (Alg concentration 0.03%) and as a negative control, DMEM medium containing 10% FBS was added 150 μL each, To prepare a cell suspension (about 100 × 10 ⁴ cells / mL).

The cell suspension was kept in a closed state at room temperature for 7 days, and a portion of the cell suspension was diluted with DMEM medium containing 10% FBS. As a reference prior to the planting density 7 days preservation was prepared cell suspension of about 10 × 10 ⁴ cells / mL. The cell suspension was inoculated in a 96-well Multiplate (manufactured by Corning Incorporated) at a rate of 100 μL, and ATP reagent (100 μL) (CellTiter-Glo ™ Luminescent Cell Viability Assay, Promega) was added thereon after 1 day and 2 days after seeding , And the mixture was allowed to stand at room temperature for 15 minutes. The emission intensity (RLU value) was measured by FlexStation 3 (manufactured by Molecular Devices) and the number of living cells was measured by subtracting the emission value of the medium alone.

As a result, the number of living cells (RLU value) of the negative control group on the day of re-sowing and the medium composition of Comparative Example 5 after 7 days of storage were significantly lowered compared with the medium compositions of Examples 2 to 4. The number of living cells (RLU value) in Example 2 and Example 4 after re-seeding increased compared to the day of re-sowing, and the cell proliferation after storage was maintained. Table 65 shows the results of the number of living cells.

[Table 65]

Industrial availability

INDUSTRIAL APPLICABILITY The culture medium composition of the present invention exhibits excellent cell and / or tissue suspension effect and is very useful when cultivating large amounts of cells and / or tissues derived from animals and plants while maintaining their functions. In addition, the cells and / or tissues cultured by the method of the present invention are useful for the evaluation of drug efficacy and toxicity of chemicals, medicines and the like, mass production of useful substances such as enzymes, cell growth factors, antibodies, Tissues, and regenerative medicine supplementing cells.

The disclosures of all publications, including the patents and patent application specifications set forth herein, are hereby incorporated by reference in their entirety to the same extent as if specifically set forth herein.

This application claims the benefit of Japanese Patent Application No. 2014-010842 (filed on January 23, 2014) and Japanese Patent Application No. 2014-123772 (filed on June 16, 2014) all of which are incorporated herein by reference. , Japanese Patent Application 2014-174574 (filed on August 28, 2014), and Japanese Patent Application 2014-217761 (filed on October 24, 2014).

Claims (27)

A medium composition capable of culturing cells or tissues in a suspension state, comprising a nanofiber. The culture medium according to claim 1, wherein the culture medium is subjected to an exchange treatment during culture, and the cells or tissue can be recovered after the culture is completed. The culture medium according to claim 1, wherein no temperature change, chemical treatment, enzyme treatment or shearing force is required at the time of cell or tissue recovery. The media composition according to claim 1, having a viscosity of 8 mPa s or less. The media composition according to claim 1, wherein the average fiber diameter of the nanofibers is 0.001 to 1.00 μm and the ratio (L / D) of the average fiber length (L) to the average fiber diameter (D) is 2 to 500. The medium composition according to claim 1, wherein the nanofiber is composed of a polymer compound. 7. The medium composition according to claim 6, wherein the polymer compound is a polysaccharide. 8. The method of claim 7,
Water-insoluble polysaccharides selected from the group consisting of cellulose, chitin and chitosan; or
There may be mentioned hyaluronic acid, gellan gum, deacylated gellan gum, rhamzaen gum, dituan gum, xanthan gum, carrageenan, xanthan gum, hexuronic acid, fucoidan, pectin, pectic acid, pectic acid, heparan sulfate, heparin, Wherein the water-soluble polysaccharide is selected from the group consisting of alginic acid, alginic acid, and salts thereof. 9. The culture medium of claim 8, wherein the polysaccharide comprises cellulose or chitin. The media composition according to claim 9, wherein the nanofibers are obtained by grinding. 11. The culture medium according to any one of claims 1 to 10, which is for cell culture. 12. The composition of claim 11, wherein the cell is an adherent cell or a non-adherent cell. 13. The media composition of claim 12, wherein the adherent cell is a sphere. 14. A cell or tissue culture comprising a culture medium according to any one of claims 1 to 13 and a cell or tissue. 14. A method for culturing a cell or tissue, comprising culturing a cell or tissue in the medium composition according to any one of claims 1 to 13. 15. A method of recovering a cell or tissue comprising separating a cell or tissue from the culture according to claim 14. 17. The method according to claim 16, wherein said separation is performed by centrifugation. 14. A method for producing a sphere, comprising culturing adherent cells in the medium composition according to any one of claims 1 to 13. 13. A culture medium additive for preparing the culture medium composition according to any one of claims 1 to 13, wherein the culture medium comprises the nanofiber or the water-soluble polymer compound constituting the nanofiber. 20. A method of producing a culture medium composition comprising mixing a culture medium with the culture medium additive according to claim 19. 14. A process for producing a medium composition according to any one of claims 1 to 13, which comprises mixing the nanofiber or the water-soluble polymer compound constituting the nanofiber with a medium. 14. A method for preserving a cell or tissue, comprising preserving the cell or tissue in the medium composition according to any one of claims 1 to 13. 14. A method of transporting a cell or tissue, comprising transporting the cell or tissue in the medium composition according to any one of claims 1 to 13. 14. A method for propagating a cell or tissue, comprising culturing the cell or tissue in the medium composition according to any one of claims 1 to 13. A subculturing method of adherent cells, comprising the steps of:
(1) suspending adherent cells in the culture composition according to any one of claims 1 to 13; And
(2) A method for culturing a cell, which comprises: (i) culturing a cell culture comprising adherent cells obtained by the suspension culture of step (1) Or (ii) fresh. In the culture medium according to any one of claims 1 to 13, all of the cultures containing the adherent cells obtained by the suspension culture of step (1) Or < / RTI > A method for propagating adherent cells comprising culturing adherent cells in a culture medium containing chitin nanofibers in a state in which adherent cells are attached to the chitin nanofibers. 27. The method according to claim 26, wherein the content of the chitin nanofibers in the culture medium composition is 0.0001% (weight / volume) to 0.1% (weight / volume).

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